Engineered nucleic acids and methods of use thereof

ABSTRACT

Provided are compositions and methods for delivering biological moieties such as modified nucleic acids into cells to modulate protein expression. Such compositions and methods include the use of modified messenger RNAs, and are useful for production of proteins.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 13/252,049, filed Oct. 3, 2011, entitled Engineered NucleicAcids and Methods of Use Thereof which claims the benefit of U.S.Provisional Application No. 61/404,413, filed Oct. 1, 2010, the contentsof each of which is incorporated herein by reference in its entirety.

REFERENCE TO THE SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitledM008USCON2SEQLST.tx created on Nov. 4, 2014 which is 57,758 bytes insize. The information in electronic format of the sequence listing isincorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

Naturally occurring RNAs are synthesized from four basicribonucleotides: ATP, CTP, UTP and GTP, but may containpost-transcriptionally modified nucleotides. Further, approximately onehundred different nucleoside modifications have been identified in RNA(Rozenski, J, Crain, P, and McCloskey, J. (1999). The RNA ModificationDatabase: 1999 update. Nucl Acids Res 27: 196-197). The role ofnucleoside modifications on the immuno-stimulatory potential and on thetranslation efficiency of RNA, however, is unclear.

There are multiple problems with prior methodologies of effectingprotein expression. For example, heterologous DNA introduced into a cellcan be inherited by daughter cells (whether or not the heterologous DNAhas integrated into the chromosome) or by offspring. Introduced DNA canintegrate into host cell genomic DNA at some frequency, resulting inalterations and/or damage to the host cell genomic DNA. In addition,multiple steps must occur before a protein is made. Once inside thecell, DNA must be transported into the nucleus where it is transcribedinto RNA. The RNA transcribed from DNA must then enter the cytoplasmwhere it is translated into protein. This need for multiple processingsteps creates lag times before the generation of a protein of interest.Further, it is difficult to obtain DNA expression in cells; frequentlyDNA enters cells but is not expressed or not expressed at reasonablerates or concentrations. This can be a particular problem when DNA isintroduced into cells such as primary cells or modified cell lines.

There is a need in the art for biological modalities to address themodulation of intracellular translation of nucleic acids.

Unless explained otherwise, all technical and scientific terms usedherein have the same meaning as commonly understood to one of ordinaryskill in the art to which this disclosure belongs. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present disclosure, suitable methods andmaterials are described herein. The materials, methods, and examples areillustrative only and not intended to be limiting. Other features of thedisclosure are apparent from the following detailed description and theclaims.

SUMMARY OF THE INVENTION

Described herein are methods of producing proteins, polypeptides, andpeptides. For example, the method includes introducing a nucleic acid(e.g., a modified nucleic acid described herein) encoding a protein,polypeptide, or peptide of interest in to a cell (e.g., a human cell),under conditions that the protein, polypeptide, or peptide of interestis produced (e.g., translated) in the cell. In some embodiments, thenucleic acid comprises one or more nucleoside modifications (e.g., oneor more nucleoside modifications described herein). In some embodiments,the nucleic acid is capable of evading an innate immune response of acell into which the nucleic acid is introduced. In some embodiments, theprotein, polypeptide, or peptide is a therapeutic protein describedherein. In some embodiments, the protein, polypeptide, or peptidecomprises one or more post-translational modifications (e.g.,post-translational modifications present in human cells). Compositionsand kits for protein production are also described herein. Furtherdescribed herein are cells and cultures with altered protein levels(e.g., generated by a method described herein).

In one aspect, the disclosure features a method of producing a protein(e.g., a heterologous protein) of interest in a cell, the methodcomprising the steps: (i) providing a target cell capable of proteintranslation; and (ii) introducing into the target cell a compositioncomprising a first isolated nucleic acid comprising a translatableregion encoding the protein of interest and a nucleoside modification,under conditions such that the protein of interest is produced in thecell In some embodiments, the method further comprises the step ofsubstantially purifying the protein of interest from the cell. In someembodiments, the protein of interest is a secreted protein.

In another aspect, the disclosure features a method of producing aprotein (e.g., a heterologous protein) of interest in a cell, the methodcomprising the steps: (i) providing a target cell capable of proteintranslation; and (ii) introducing into the target cell a compositioncomprising: (a) a first isolated nucleic acid comprising a translatableregion encoding the protein of interest and a nucleoside modification;and (b) a second nucleic acid comprising an inhibitory nucleic acid,under conditions such that the protein of interest is produced in thecell. In some embodiments, the method further comprises the step ofsubstantially purifying the protein of interest from the cell. In someembodiments, the protein of interest is a secreted protein.

In one aspect, the disclosure features a method of increasing theproduction of a recombinantly expressed protein of interest in a cell,comprising the steps: (i) providing a target cell comprising arecombinant nucleic acid encoding the protein of interest; and (ii)introducing into the target cell a composition comprising a firstisolated nucleic acid comprising a translatable region encoding atranslation effector protein and a nucleoside modification underconditions such that the effector protein is produced in the cell,thereby increasing the production of the recombinantly expressed proteinin the cell.

In some embodiments, the target cell is a mammalian cell. In someembodiments, the target cell is a yeast cell. In some embodiments, thetarget cell is a bacterial cell, an insect cell, or a plant cell. Insome embodiments, the protein of interest is a secreted protein. In someembodiments, the protein of interest is a transmembrane protein. In someembodiments, the protein of interest is an antibody or anantigen-binding fragment thereof. In some embodiments, the protein ofinterest is a growth factor or cytokine. In some embodiments, theprotein of interest is a peptide or peptidomimetic. In some embodiments,the translation effector protein is ceramide transfer protein (CERT). Insome embodiments, the translation effector protein is translated in thetarget cell in an amount effective to increase efficiency of translationof the recombinantly expressed protein. In some embodiments, thetranslation effector protein is translated in the target cell in anamount effective to reduce efficiency of translation of proteins in thecell other than the recombinantly expressed protein. In someembodiments, the translation effector protein is translated in thetarget cell in an amount effective to reduce formation of inclusionbodies containing the recombinantly expressed protein. In someembodiments, the translation effector protein is translated in thetarget cell in an amount effective to reduce intracellular degradationof the recombinantly expressed protein. In some embodiments, thetranslation effector protein is translated in the target cell in anamount effective to increase secretion of the recombinantly expressedprotein.

In another aspect, the disclosure features a method for altering thelevel of a protein of interest in a target cell, the method comprisingthe steps of: (i) modulating the activity of at least one translationeffector molecule in the target cell; and (ii) culturing the cell. Insome embodiments, the target cell does not contain a recombinant nucleicacid. In some embodiments, the method further comprises the step ofisolating the protein of interest.

In another aspect, the disclosure features a method for modulating thelevel of a protein of interest in a target cell, comprising the stepsof: i) modulating the activity of at least one translation effectormolecule in the target cell, wherein the modulation comprisesintroducing into the target cell a first isolated nucleic acidcomprising a translatable region encoding the translation effectorprotein and a nucleoside modification; and ii) culturing the cell.

In one aspect, the disclosure features an animal cell (e.g., a mammaliancell) with an altered protein level, generated by the steps of: (i)introducing into the cell an effective amount of a first isolatednucleic acid comprising a translatable region encoding a translationeffector protein and a nucleoside modification; and (ii) culturing thecell. In some embodiments, the effective amount of the first isolatednucleic acid introduced into the cell is titrated against a desiredamount of protein translated from the translatable region.

In one aspect, the disclosure features a high density culture comprisinga plurality of the cells described herein. In some embodiments, theculture comprises a batch process. In some embodiments, the culturecomprises a continuous feed process.

In one aspect, the disclosure features a composition for proteinproduction, the composition comprising a first isolated nucleic acidcomprising a translatable region and a nucleoside modification, whereinthe nucleic acid exhibits reduced degradation by a cellular nuclease,and a mammalian cell suitable for translation of the translatable regionof the first nucleic acid. In some embodiments, the mammalian cellcomprises a recombinant nucleic acid.

In another aspect, the disclosure features a composition for proteinproduction, the composition comprising: (i) a first isolated nucleicacid comprising a translatable region and a nucleoside modification,wherein the nucleic acid exhibits reduced degradation by a cellularnuclease; (ii) a second nucleic acid comprising an inhibitory nucleicacid; and (iii) a mammalian cell suitable for translation of thetranslatable region of the first nucleic acid, wherein the mammaliancell comprises a target nucleic acid capable of being acted upon by theinhibitory nucleic acid. In some embodiments, the mammalian cellcomprises a recombinant nucleic acid.

In one aspect, the disclosure features a kit for protein production, thekit comprising a first isolated nucleic acid comprising a translatableregion and a nucleic acid modification, wherein the nucleic acid iscapable of evading an innate immune response of a cell into which thefirst isolated nucleic acid is introduced, and packaging andinstructions therefor.

In another aspect, the disclosure features a kit for protein production,the kit comprising: (i) a first isolated nucleic acid comprising atranslatable region, provided in an amount effective to produce adesired amount of a protein encoded by the translatable region whenintroduced into a target cell; (ii) a second nucleic acid comprising aninhibitory nucleic acid, provided in an amount effective tosubstantially inhibit the innate immune response of the cell; and (iii)packaging and instructions therefor.

In yet another aspect, the disclosure features a kit for proteinproduction, the kit comprising a first isolated nucleic acid comprisinga translatable region and a nucleoside modification, wherein the nucleicacid exhibits reduced degradation by a cellular nuclease, and packagingand instructions therefor.

In one aspect, the disclosure features a kit for protein production, thekit comprising a first isolated nucleic acid comprising a translatableregion and at least two different nucleoside modifications, wherein thenucleic acid exhibits reduced degradation by a cellular nuclease, andpackaging and instructions therefor.

In another aspect, the disclosure features a kit for protein production,the kit comprising: (i) a first isolated nucleic acid comprising atranslatable region; (ii) a second nucleic acid comprising an inhibitorynucleic acid; and (iii) packaging and instructions therefor.

In yet another aspect, the disclosure features a kit for proteinproduction, the kit comprising: (i) a first isolated nucleic acidcomprising a translatable region and at least one nucleosidemodification, wherein the nucleic acid exhibits reduced degradation by acellular nuclease; (ii) a second nucleic acid comprising an inhibitorynucleic acid; and (iii) packaging and instructions therefor.

In one aspect, the disclosure features a kit for protein production,comprising a first isolated nucleic acid encoding a translatable regionencoding a protein, wherein the first nucleic acid comprises a nucleicacid modification, wherein the first nucleic acid displays decreaseddegradation in a cell into which the first isolated nucleic acid isintroduced as compared to a nucleic acid not comprising a nucleic acidmodification, and packaging and instructions therefor.

In another aspect, the disclosure features a kit for protein production,comprising a first isolated nucleic acid encoding a translatable regionencoding a protein, wherein the first nucleic acid comprises a nucleicacid modification, wherein the first nucleic acid displays is morestable in a cell into which the first isolated nucleic acid isintroduced as compared to a nucleic acid not comprising a nucleic acidmodification, and packaging and instructions therefor.

In one aspect, the disclosure features a kit for immunoglobulin proteinproduction, comprising a first isolated nucleic acid comprising i) atranslatable region encoding the immunoglobulin protein and ii) anucleic acid modification, wherein the first nucleic acid is capable ofevading an innate immune response of a cell into which the firstisolated nucleic acid is introduced, wherein the translatable region issubstantially devoid of cytidine and uracil nucleotides, and packagingand instructions therefor.

In another aspect, the disclosure features a mammalian cell generated byuse of a kit described herein.

In yet another aspect, the disclosure features an isolatedimmunoglobulin protein produced from a production cell comprising afirst isolated nucleic acid comprising i) a translatable region encodingthe immunoglobulin protein and ii) a nucleic acid modification, whereinthe first nucleic acid is capable of evading an innate immune responseof the cell, wherein the translatable region is substantially devoid ofeither cytidine or uracil nucleotides or the combination of cytidine anduracil nucleotides.

In one aspect, the disclosure features a pharmaceutical preparationcomprising an effective amount of a protein described herein.

In another aspect, the disclosure features a pharmaceutical preparationcomprising an effective amount of a first nucleic acid comprising i) atranslatable region encoding an immunoglobulin protein and ii) a nucleicacid modification, wherein the first nucleic acid exhibits reduceddegradation by a cellular nuclease and is capable of evading an innateimmune response of a cell into which the first nucleic acid isintroduced, wherein the translatable region is substantially devoid ofcytidine and uracil nucleotides.

Embodiments of the aforesaid methods, cells, cultures, compositions,preparations, and kits may include one or more of the followingfeatures:

In some embodiments, the first isolated nucleic acid comprises messengerRNA (mRNA). In some embodiments, the mRNA comprises at least onenucleoside selected from the group consisting of pyiidin-4-oneribonucleoside, 5-aza-uridine, 2-thio-5-aza-midine, 2-thiouridine,4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine,3-methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine,5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyluridine,1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine,1-taulinomethyl-4-thio-uridine, 5-methyl-uridine,1-methyl-pseudouridine, 4-thio-1-methyl-pseudouridine,2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine,2-thio-1-methyl-1-deaza-pseudomidine, dihydrouridine,dihydropseudouridine, 2-thio-dihydromidine, 2-thio-dihydropseudouridine,2-methoxyuridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, and4-methoxy-2-thio-pseudouridine. In some embodiments, the mRNA comprisesat least one nucleoside selected from the group consisting of5-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine,5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine,1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine,2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine,4-thio-1-methyl-pseudoisocytidine,4-thio-1-methyl-1-deaza-pseudoisocytidine,1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine,5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine,2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine,4-methoxy-pseudoisocytidine, and 4-methoxy-1-methyl-pseudoisocytidine.In some embodiments, the mRNA comprises at least one nucleoside selectedfrom the group consisting of 2-aminopurine, 2,6-diaminopurine,7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine,7-deaza-8-aza-2-aminopurine, 7-deaza-2,6-diarminopurine,7-deaza-8-aza-2,6-diarninopurine, 1-methyladenosine, N6-methyladenosine,N6-isopentenyladenosine, N6-(cis-hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl) adenosine,N6-glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine,2-methylthio-N6-threonyl carbamoyladenosine, N6,N6-dimethyladenosine,7-methyladenine, 2-methylthio-adenine, and 2-methoxy-adenine. In someembodiments, mRNA comprises at least one nucleoside selected from thegroup consisting of inosine, 1-methyl-inosine, wyosine, wybutosine,7-deaza-guanosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine,6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine,7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine,6-methoxy-guanosine, 1-methylguanosine, N2-methylguanosine,N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine,1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, andN2,N2-dimethyl-6-thio-guanosine.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts bar graphs of an Enzyme-linked immunosorbent assay(ELISA) detection of Human G-CSF of in vitro transfected Chinese HamsterOvary with modRNA encoding human G-CSF at 12 and 24 hourspost-transfection.

FIG. 2 depicts bar graphs of an Enzyme-linked immunosorbent assay(ELISA) for Human IgG of in vitro transfected Chinese Hamster Ovarycells with the Heavy and Light chains of modRNA encoding Trastuzumab at12, 24, and 36 hours post-transfection.

FIG. 3 depicts bar graphs of an Enzyme-linked immunosorbent assay(ELISA) for detection of Human IgG of in vitro transfected HumanEmbryonic Kidneys cells (HEK293) with Heavy and Light chains of modRNAencoding Trastuzumab at 36 hours post-transfection. R1, R2, R3 aretriplicate transfection experiments performed in a 24-well plate andnormalized to untreated samples.

FIG. 4 depicts an image of a western blot detection of in vitrotransfected Chinese Hamster Ovary cells with the Heavy and Light chainsof modRNA encoding Trastuzumab at 24 hours post-transfection. HC and LCindicate the Heavy Chain and Light Chain of Trastuzumab respectively.

FIG. 5 depicts images from cell immune-staining of in vitro-transfectedChinese Hamster Ovary cells with the Heavy and Light chains of modRNAencoding both Trastuzumab and Rituximab at 13 hours post-transfection.

FIG. 6 depicts images of a binding immunoblot assay of modRNA encodingTrastuzumab and Rituximab. The black boxes display the protein ofinterest.

DETAILED DESCRIPTION OF THE INVENTION

Methods of producing proteins, polypeptides, and peptides are describedherein. The disclosure provides, at least in pmt, methods of producing aprotein, polypeptide, or peptide (e.g., a heterologous protein) ofinterest in a cell, methods increasing the production of a protein,polypeptide, or peptide (e.g., a recombinantly expressed protein) ofinterest in a cell, and methods of altering the level of a protein,polypeptide, or peptide of interest in a cell. For example, the methodscan include the step of introducing a nucleic acid (e.g., a modifiednucleic acid described herein) encoding a protein, polypeptide, orpeptide of interest into a cell (e.g., a human cell), under conditionsthat the protein, polypeptide, or peptide of interest is produced (e.g.,translated) in the cell. In some embodiments, the nucleic acid comprisesone or more nucleoside modifications (e.g., one or more nucleosidemodifications described herein). In some embodiments, the nucleic acidis capable of evading an innate immune response of a cell into which thenucleic acid is introduced, thus increasing the efficiency of proteinproduction in the cell. In some embodiments, the protein is atherapeutic protein described herein. In some embodiments, the proteincomprises one or more post-translational modifications (e.g.,post-translational modifications present in human cells). Compositionsand kits for protein production are also described herein. Furtherdescribed herein are cells and cultures with altered protein levels(e.g., generated by a method described herein).

In general, exogenous nucleic acids, particularly viral nucleic acids,introduced into cells induce an innate immune response, resulting ininterferon (IFN) production and cell death. However, it is of greatinterest for recombinant protein production to deliver a nucleic acid,e.g., a ribonucleic acid (RNA) inside a cell, e.g., in cell culture, invitro, in vivo, or ex vivo, such as to cause intracelluar translation ofthe nucleic acid and production of the encoded protein. Provided hereinin part are nucleic acids encoding useful polypeptides capable ofmodulating a cell's function and/or activity, and methods of making andusing these nucleic acids and polypeptides. As described herein, thesenucleic acids are capable of reducing the innate immune activity of apopulation of cells into which they are introduced, thus increasing theefficiency of protein production in that cell population. Further, oneor more additional advantageous activities and/or properties of thenucleic acids and proteins of the invention are described.

Methods of Protein Production.

The methods provided herein are useful for enhancing protein productyield in a cell culture process. In a cell culture containing aplurality of host cells, introduction of the modified mRNAs describedherein results in increased protein production efficiency relative to acorresponding unmodified nucleic acid. Such increased protein productionefficiency can be demonstrated, e.g., by showing increased celltransfection, increased protein translation from the nucleic acid,decreased nucleic acid degradation, and/or reduced innate immuneresponse of the host cell. Protein production can be measured by ELISA,and protein activity can be measured by various functional assays knownin the mt. The protein production may be generated in a continuous or afed-batch process.

Cell Culture and Growth.

In the methods of the disclosure, the cells are cultured. Cells may becultured in suspension or as adherent cultures. Cells may be cultured ina variety of vessels including, for example, bioreactors, cell bags,wave bags, culture plates, flasks, hyperflasks and other vessels wellknown to those of ordinary skill in the art. Cells may be cultured inIMDM (Invitrogen, Catalog number 12440-53) or any other suitable mediaincluding chemically defined media formulations. Ambient conditionssuitable for cell culture, such as temperature and atmosphericcomposition, are also well known to those skilled in the art. Themethods of the disclosure may be used with any cell that is suitable foruse in protein production. In one embodiment, the cells are selectedfrom the group consisting of animal cells (e.g., mammalian cells),bacterial cells, plant, microbial, algal, and fungal cells. In someembodiments, the cells are mammalian cells, such human, mouse, rat,goat, horse, rabbit, hamster or cow cells. For instance, the cells maybe from an y established cell line, including but not limited to HeLa,NSO, SP2/0, HEK 293T, Vero, Caco, Caco-2, MDCK, COS-1, COS-7, K562,Jurkat, CHO-K1, DG44, CHOK1SV, CHO-S, Huvec, CV-1, HuH-7, NIH3T3,HEK293, 293, A549, HepG2, IMR-90, MCF-7, U-205, Per.C6, SF9, SF21, orChinese Hamster Ovary (CHO) cells. In certain embodiments, the cells arefungal cells, such as cells selected from the group consisting of:Chrysosporium cells, Aspergillus cells, Trichoderma cells, Dictyosteliumcells, Candida cells, Saccharomyces cells, Scbizosaccharomyces cells,and Penicillium cells. In certain other embodiments, the cells arebacterial cells, such as E. coli, B. subtilis, or BL21 cells. Primaryand secondary cells to be transfected by the present method can beobtained from a variety of tissues and include all cell types which canbe maintained in culture. For example, primary and secondary cells whichcan be transfected by the present method include fibroblasts,keratinocytes, epithelial cells (e.g., mammary epithelial cells,intestinal epithelial cells), endothelial cells, glial cells, neuralcells, formed elements of the blood (e.g., lymphocytes, bone marrowcells), muscle cells and precursors of these somatic cell types. Primarycells can be obtained from a donor of the same species or anotherspecies (e.g., mouse, rat, rabbit, cat, dog, pig, cow, bird, sheep,goat, horse).

The cells of the present disclosure are useful for in vitro productionof therapeutic products which can be purified and delivered byconventional routes of administration. With or without amplification,these cells can be subject to large-scale cultivation for harvest ofintracellular or extracellular protein products.

Methods of Cellular Nucleic Acid Delivery.

Methods of the present disclosure enhance nucleic acid delivery into acell population, in vivo, ex vivo, or in culture. For example, a cellculture containing a plurality of host cells (e.g., eukaryotic cellssuch as yeast or mammalian cells) is contacted with a composition thatcontains an enhanced nucleic acid having at least one nucleosidemodification and, optionally, a translatable region. The compositionalso generally contains a transfection reagent or other compound thatincreases the efficiency of enhanced nucleic acid uptake into the hostcells. The enhanced nucleic acid exhibits enhanced retention in the cellpopulation, relative to a corresponding unmodified nucleic acid. Theretention of the enhanced nucleic acid is greater than the retention ofthe unmodified nucleic acid. In some embodiments, it is at least about50%, 75%, 90%, 95%, 100%, 150%, 200%, or more than 200% greater than theretention of the unmodified nucleic acid. Such retention advantage maybe achieved by one round of transfection with the enhanced nucleic acid,or may be obtained following repeated rounds of transfection.

Introduction of Modified or Transient RNAs into Cells for ProteinProduction.

Transiently transfected cells may be generated by methods oftransfection, electroporation, cationic agents, polymers, or lipid-baseddelivery molecules well known to those of ordinary skill in the art. Themodified transient RNAs can be introduced into the cultured cells ineither traditional batch like steps or continuous flow through steps ifappropriate. The methods and compositions of the present disclosure maybe used to produce cells with increased production of one or moreprotein of interest. Cells can be transfected or otherwise introducedwith one or more RNA. The cells may be transfected with the two or moreRNA constructs simultaneously or sequentially. In certain embodiments,multiple rounds of the methods described herein may be used to obtaincells with increased expression of one or more RNAs or proteins ofinterest. For example, cells may be transfected with one or more RNAconstructs that encode an RNA or protein of interest and isolatedaccording to the methods described herein. The isolated cells may thenbe subjected to further rounds of transfection with one or more otherRNA that encode an RNA or protein of interest and isolated once again.This method is useful, for example, for generating cells with increasedexpression of a complex of proteins, RNAs or proteins in the same orrelated biological pathway, RNAs or proteins that act upstream ordownstream of each other, RNAs or proteins that have a modulating,activating or repressing function to each other, RNAs or proteins thatare dependent on each other for function or activity, or RNAs orproteins that share homology (e.g., sequence, structural, or functionalhomology). For example, this method may be used to generate a cell linewith increased expression of the heavy and light chains of animmunoglobulin protein (e.g., IgA, IgD, IgE, IgG, and IgM) orantigen-binding fragments thereof. The immunoglobulin proteins may befully human, humanized, or chimeric immunoglobulin proteins. An RNA thatis transfected into a cell of the disclosure may comprise a sequencethat is an RNA encoding a protein of interest. Any protein may beproduced according to the methods described herein. Examples of proteinsthat may be produced according the methods of the disclosure include,without limitation, peptide hormones (e.g., insulin), glycoproteinhormones (e.g., erythropoietin), antibiotics, cytokines, enzymes,vaccines (e.g., HIV vaccine, HPV vaccine, HBV vaccine), anticancertherapeutics (e.g., Muc1), and therapeutic antibodies. In a particularembodiment the RNA encodes an immunoglobulin protein or anantigen-binding fragment thereof, such as an immunoglobulin heavy chain,an immunoglobulin light chain, a single chain Fv, a fragment of anantibody, such as Fab, Fab′, or (Fab)₂, or an antigen binding fragmentof an immunoglobulin. In a specific embodiment, the RNA encodeserythropoietin. In another specific embodiment, the RNA encodes one ormore immunoglobulin proteins, or fragments thereof, that bind to and,optionally, antagonize or agonize a cell surface receptor: the epidermalgrowth factor receptor (EGFR), HER2, or c-ErbB-1, such as Erbitux™(cetuximab).

Isolation or Purification of Proteins.

The methods described herein can further comprise the step of isolatingor purifying the proteins, polypeptides, or peptides produced by themethods described herein. Those of ordinary skill in the art can easilymake a determination of the proper manner to purify or isolate theprotein of interest from the cultured cells. Generally, this is donethrough a capture method using affinity binding or non-affinitypurification. If the protein of interest is not secreted by the culturedcells, then a lysis of the cultured cells would be performed prior topurification or isolation as described above. One can use unclarifiedcell culture fluid containing the protein of interest along with cellculture media components as well as cell culture additives, such asanti-foam compounds and other nutrients and supplements, cells, cellulardebris, host cell proteins, DNA, viruses and the like in the presentdisclosure. Moreover, the process can be conducted, if desired, in thebioreactor itself. The fluid may either be preconditioned to a desiredstimulus such as pH, temperature or other stimulus characteristic or thefluid can be conditioned upon addition of the polymer(s) or thepolymer(s) can be added to a carrier liquid that is properly conditionedto the required parameter for the stimulus condition required for thatpolymer to be solubilized in the fluid. The polymer(s) is allowed tocirculate thoroughly with the fluid and then the stimulus is applied(change in pH, temperature, salt concentration, etc) and the desiredprotein and polymer(s) precipitate out of solution. The polymer anddesired protein(s) is separated from the rest of the fluid andoptionally washed one or more times to remove any trapped or looselybound contaminants. The desired protein is then recovered from thepolymer(s) such as by elution and the like. Typically, the elution isdone under a set of conditions such that the polymer remains in itssolid (precipitated) form and retains any impurities to it during theselective elution of the desired protein. Alternatively, the polymer andprotein as well as any impurities can be solubilized in a new fluid suchas water or a buffered solution and the protein be recovered by a meanssuch as affinity, ion exchange, hydrophobic, or some other type ofchromatography that has a preference and selectivity for the proteinover that of the polymer or impurities. The eluted protein is thenrecovered and if desired subjected to additional processing steps,either traditional batch like steps or continuous flow through steps ifappropriate.

Additionally, it is useful to optimize the expression of a specificpolypeptide in a cell line or collection of cell lines of potentialinterest, particularly an engineered protein such as a protein variantof a reference protein having a known activity. In one embodiment,provided is a method of optimizing expression of an engineered proteinin a target cell, by providing a plurality of target cell types, andindependently contacting with each of the plurality of target cell typesa modified mRNA encoding an engineered polypeptide. Additionally,culture conditions may be altered to increase protein productionefficiency. Subsequently, the presence and/or level of the engineeredpolypeptide in the plurality of target cell types is detected and/orquantitated, allowing for the optimization of an engineeredpolypeptide's expression by selection of an efficient target cell andcell culture conditions relating thereto. Such methods are particularlyuseful when the engineered polypeptide contains one or morepost-translational modifications or has substantial tertiary structure,situations which often complicate efficient protein production.

“Proteins of interest” or “desired proteins” include those providedherein and fragments, mutants, variants, and alterations thereof.Especially, desired proteins/polypeptides or proteins of interest arefor example, but not limited to insulin, insulin-like growth factor,human growth hormone (hGH), tissue plasminogen activator (tPA),cytokines, such as interleukins (IL), e.g., IL-1, IL-2, IL-3, IL-4,IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15,IL-16, IL-17, IL-18, interferon (IFN) alpha, IFN beta, IFN gamma, IFNomega or IFN tau, tumor necrosis factor (TNF), such as TNF alpha and TNFbeta, TNF gamma, TNF-related apoptosis-inducing ligand (TRAIL);granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophagecolony-stimulating factor (GM-CSF), macrophage colony-stimulating factor(M-CSF), monocyte chemotactic protein-1 (MCP-1), and vascularendothelial growth factor (VEGF). Also included is the production oferythropoietin or any other hormone growth factors. The method accordingto the disclosure can also be advantageously used for production ofantibodies or fragments thereof. Such fragments include e.g., Fabfragments (Fragment antigen-binding). Fab fragments consist of thevariable regions of both chains which are held together by the adjacentconstant region. These may be formed by protease digestion, e.g., withpapain, from conventional antibodies, but similar Fab fragments may alsobe produced in the mean time by genetic engineering. Further antibodyfragments include F(ab′)2 fragments, which may be prepared byproteolytic cleaving with pepsin.

The protein of interest is typically recovered from the culture mediumas a secreted polypeptide, or it can be recovered from host cell lysatesif expressed without a secretory signal. It is necessary to purify theprotein of interest from other recombinant proteins and host cellproteins in a way that substantially homogenous preparations of theprotein of interest are obtained. As a first step, cells and/orparticulate cell debris are removed from the culture medium or lysate.The product of interest thereafter is purified from contaminant solubleproteins, polypeptides and nucleic acids, for example, by fractionationon immunoaffinity or ion-exchange columns, ethanol precipitation,reverse phase HPLC, Sephadex chromatography, chromatography on silica oron a cation exchange resin such as DEAE. In general, methods teaching askilled person how to purify a protein heterologous expressed by hostcells, are well known in the art. Such methods are for example describedby (Harris and Angal, Protein Purification Methods: A PracticalApproach, Oxford University Press, 1995) or (Robert Scopes, ProteinPurification: Principles and Practice, Springer, 1988).

Methods of the present disclosure enhance nucleic acid delivery into acell population, in vivo, ex vivo, or in culture. For example, a cellculture containing a plurality of host cells (e.g., eukaryotic cellssuch as yeast or mammalian cells) is contacted with a composition thatcontains an enhanced nucleic acid having at least one nucleosidemodification and, optionally, a translatable region. The compositionalso generally contains a transfection reagent or other compound thatincreases the efficiency of enhanced nucleic acid uptake into the hostcells. The enhanced nucleic acid exhibits enhanced retention in the cellpopulation, relative to a corresponding unmodified nucleic acid. Theretention of the enhanced nucleic acid is greater than the retention ofthe unmodified nucleic acid. In some embodiments, it is at least about50%, 75%, 90%, 95%, 100%, 150%, 200%, or more than 200% greater than theretention of the unmodified nucleic acid. Such retention advantage maybe achieved by one round of transfection with the enhanced nucleic acid,or may be obtained following repeated rounds of transfection.

In some embodiments, the enhanced nucleic acid is delivered to a targetcell population with one or more additional nucleic acids. Such deliverymay be at the same time, or the enhanced nucleic acid is delivered priorto delivery of the one or more additional nucleic acids. The additionalone or more nucleic acids may be modified nucleic acids or unmodifiednucleic acids. It is understood that the initial presence of theenhanced nucleic acids does not substantially induce an innate immuneresponse of the cell population and, moreover, that the innate immuneresponse will not be activated by the later presence of the unmodifiednucleic acids. In this regard, the enhanced nucleic acid may not itselfcontain a translatable region, if the protein desired to be present inthe target cell population is translated from the unmodified nucleicacids.

Antagonist Protein Expression.

Methods and compositions described herein can be used to producedproteins that are capable of attenuating or blocking the endogenousagonist biological response and/or antagonizing a receptor or signalingmolecule in a mammalian subject. For example, IL-12 and IL-23 receptorsignaling is enhanced in chronic autoimmune disorders such as multiplesclerosis and inflammatory diseases such as rheumatoid arthritis,psoriasis, lupus erythematosus, ankylosing spondylitis and Crohn'sdisease (Kikly K, Liu L, Na S, Sedgwick J D (2006) Curr. Opin. Immunol.18 (6): 670-5). In another embodiment, a nucleic acid encodes anantagonist for chemokine receptors. Chemokine receptors CXCR-4 and CCR-5are required for HIV entry into host cells (Arenzana-Seisdedos F et al,(1996) Nature. October 3; 383 (6599):400).

Targeting Moieties.

In embodiments of the disclosure, modified nucleic acids are provided toexpress a protein-binding partner or a receptor on the surface of thecell, which functions to target the cell to a specific tissue space orto interact with a specific moiety, either in vivo or in vitro. Suitableprotein-binding partners include antibodies and functional fragmentsthereof, scaffold proteins, or peptides. Additionally, modified nucleicacids can be employed to direct the synthesis and extracellularlocalization of lipids, carbohydrates, or other biological moieties.

Permanent Gene Expression Silencing.

A method for epigenetically silencing gene expression in a mammaliansubject, comprising a nucleic acid where the translatable region encodesa polypeptide or polypeptides capable of directing sequence-specifichistone H3 methylation to initiate heterochromatin formation and reducegene transcription around specific genes for the purpose of silencingthe gene. For example, a gain-of-function mutation in the Janus Kinase 2gene is responsible for the family of Myeloproliferative Diseases.

Mechanism Details.

Fission yeast require two RNAi complexes for siRNA-mediatedheterochromatin assembly: the RNA-induced transcriptional silencing(RITS) complex and the RNA-directed RNA polymerase complex (RDRC)(Motamedi et al. Cell 2004, 119, 789-802). In fission yeast, the RITScomplex contains the siRNA binding Argonaute family protein Ago1, achromodomain protein Chp1, and Tas3. The fission yeast RDRC complex iscomposed of an RNA-dependent RNA Polymerase Rdp1, a putative RNAhelicase Hrr1, and a polyA polymerase family protein Cid12. These twocomplexes require the Dicer ribonuclease and Clr4 histone H3methyltransferase for activity. Together, Ago1 binds siRNA moleculesgenerated through Dicer-mediated cleavage of Rdp1 co-transcriptionallygenerated dsRNA transcripts and allows for the sequence-specific directassociation of Chp1, Tas3, Hrr1, and Clr4 to regions of DNA destined formethylation and histone modification and subsequent compaction intotranscriptionally silenced heterochromatin. While this mechanismfunctions in cis- with centromeric regions of DNA, sequence-specifictrans silencing is possible through co-transfection with double-strandedsiRNAs for specific regions of DNA and concomitant RNAi-directedsilencing of the siRNA ribonuclease Elil (Buhler et al. Cell 2006, 125,873-886).

Production of Polypeptide Variants.

Methods and compositions described herein can be used for production ofpolypeptide variants. Provided herein are nucleic acids that encodevariant polypeptides, which have a certain identity with a referencepolypeptide sequence. The term “identity” as known in the art, refers toa relationship between the sequences of two or more peptides, asdetermined by comparing the sequences. In the art, “identity” also meansthe degree of sequence relatedness between peptides, as determined bythe number of matches between strings of two or more amino acidresidues. “Identity” measures the percent of identical matches betweenthe smaller of two or more sequences with gap alignments (if any)addressed by a particular mathematical model or computer program (i.e.,“algorithms”). Identity of related peptides can be readily calculated byknown methods. Such methods include, but are not limited to, thosedescribed in Computational Molecular Biology, Lesk, A. M., ed., OxfordUniversity Press, New York, 1988; Biocomputing: Informatics and GenomeProjects, Smith, D. W., ed., Academic Press, New York, 1993; ComputerAnalysis of Sequence Data, Prut 1, Griffin, A. M., and Gtiffin, H. G.,eds., Humana Press, New Jersey, 1994; Sequence Analysis in MolecularBiology, von Heinje, G., Academic Press, 1987; Sequence Analysis Primer,Gribskov, M. and Devereux, J., eds., M. Stockton Press, New York, 1991;and Calillo et al., SIAM J. Applied Math. 48, 1073 (1988).

In some embodiments, the polypeptide variant has the same or a similaractivity as the reference polypeptide. Alternatively, the variant has analtered activity (e.g., increased or decreased) relative to a referencepolypeptide. Generally, variants of a particular polynucleotide orpolypeptide of the disclosure will have at least about 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or more sequence identity to that particular referencepolynucleotide or polypeptide as determined by sequence alignmentprograms and parameters described herein and known to those skilled inthe art.

As recognized by those skilled in the art, protein fragments, functionalprotein domains, and homologous proteins are also considered to bewithin the scope of this disclosure. For example, provided herein is anyprotein fragment of a reference protein (meaning a polypeptide sequenceat least one amino acid residue shorter than a reference polypeptidesequence but otherwise identical) about 10, 20, 30, 40, 50, 60, 70, 80,90, 100, or greater than 100 amino acids in length. In another example,any protein that includes a stretch of about 20, about 30, about 40,about 50, or about 100 amino acids, which are about 40%, about 50%,about 60%, about 70%, about 80%, about 90%, about 95%, or about 100%identical to any of the sequences described herein, can be utilized inaccordance with the disclosure. In certain embodiments, a proteinsequence to be utilized in accordance with the disclosure includes 2, 3,4, 5, 6, 7, 8, 9, 10, or more mutations as shown in any of the sequencesprovided or referenced herein.

Production of Polypeptide Libraries

Methods and compositions described herein can be used for production ofpolypeptide libraries. Provided herein are polynucleotide librariescontaining nucleoside modifications, wherein the polynucleotidesindividually contain a first nucleic acid sequence encoding apolypeptide, such as an antibody, protein binding partner, scaffoldprotein, and other polypeptides known in the art. Typically, thepolynucleotides are mRNA in a form suitable for direct introduction intoa target cell host, which in turn synthesizes the encoded polypeptide.

In certain embodiments, multiple variants of a protein, each withdifferent amino acid modification(s), are produced and tested todetermine the best valiant in terms of pharmacokinetics, stability,biocompatibility, and/or biological activity, or a biophysical propertysuch as expression level. Such a library may contain about 10, 10², 10³,10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, or over 10⁹ possible variants (includingsubstitutions, deletions of one or more residues, and insertion of oneor more residues).

Production of Polypeptide-Nucleic Acid Complexes

Methods and compositions described herein can be used for production ofpolypeptide-nucleic acid complexes. Proper protein translation involvesthe physical aggregation of a number of polypeptides and nucleic acidsassociated with the rnRNA. Provided by the disclosure areprotein-nucleic acid complexes, containing a translatable mRNA havingone or more nucleoside modifications (e.g., at least two differentnucleoside modifications) and one or more polypeptides bound to themRNA. Generally, the proteins are provided in an amount effective toprevent or reduce an innate immune response of a cell into which thecomplex is introduced.

Production of Untranslatable Modified Nucleic Acids.

Methods and compositions described herein can be used for production ofuntranslatable modified nucleic acids. As described herein, provided aremRNAs having sequences that are substantially not translatable. SuchmRNA is effective as a vaccine when administered to a mammalian subject.

Also provided are modified nucleic acids that contain one or morenoncoding regions. Such modified nucleic acids are generally nottranslated, but are capable of binding to and sequestering one or moretranslational machinery component such as a ribosomal protein or atransfer RNA (tRNA), thereby effectively reducing protein expression inthe cell. The modified nucleic acid may contain a small nucleolar RNA(sno-RNA), microRNA (miRNA), small interfering RNA (sRNA), small hairpinRNA (shRNA), or Piwi-interacting RNA (piRNA).

Modified Nucleic Acids.

This disclosure provides methods of producing proteins using nucleicacids, including RNAs such as messenger RNAs (mRNAs) that contain one ormore modified nucleosides (termed “modified nucleic acids”), which haveuseful properties including the lack of a substantial induction of theinnate immune response of a cell into which the mRNA is introduced.Because these modified nucleic acids enhance the efficiency of proteinproduction, intracellular retention of nucleic acids, and viability ofcontacted cells, as well as possess reduced immunogenicity, thesenucleic acids having these properties are termed “enhanced nucleicacids” herein.

The term “nucleic acid,” in its broadest sense, includes any compoundand/or substance that is or can be incorporated into an oligonucleotidechain. Exemplary nucleic acids for use in accordance with the presentdisclosure include, but are not limited to, one or more of DNA, RNAincluding messenger mRNA (mRNA), hybrids thereof, RNA interference(RNAi)-inducing agents, RNAi agents, small interfering RNAs (siRNAs),small hairpin RNAs (shRNAs), microRNAs (miRNAs), antisense RNAs,ribozymes, catalytic DNA, RNAs that induce triple helix formation,aptamers, vectors, etc., described in detail herein.

Provided are modified nucleic acids containing a translatable region andone, two, or more than two different nucleoside modifications. In someembodiments, the modified nucleic acid exhibits reduced degradation in acell into which the nucleic acid is introduced, relative to acorresponding unmodified nucleic acid. For example, the degradation rateof the modified nucleic acid is reduced by about 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90%, or greater than 90%, compared to thedegradation rate of the corresponding unmodified nucleic acid. Exemplarynucleic acids include ribonucleic acids (RNAs), deoxyribonucleic acids(DNAs), threose nucleic acids (TNAs), glycol nucleic acids (GNAs), or ahybrid thereof. In typical embodiments, the modified nucleic acidincludes messenger RNAs (mRNAs). As described herein, the nucleic acidsof the disclosure do not substantially induce an innate immune responseof a cell into which the mRNA is introduced.

In some embodiments, modified nucleosides include pyridin-4-oneribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiomidine,4-thio-pseudomidine, 2-thio-pseudowidine, 5-hydroxyuridine,3-methylmidine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudoutidine,5-propynyl-uridine, 1-propynyl-pseudomidine, 5-taurinomethyluridine,1-taurinomethyl-pseudouridine, 5-taw.inomethyl-2-thio-utidine,1-taurinomethyl-4-thio-uridine, 5-methyl-uridine,1-methyl-pseudouridine, 4-thio-1-methyl-pseudouridine,2-thio-1-methyl-pseudoutidine, 1-methyl-1-deaza-pseudomidine,2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine,dihydropseudouridine, 2-thio-dihydromidine, 2-thio-dihydropseudoulidine,2-methoxyuridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudomidine, and4-methoxy-2-thio-pseudouridine.

In some embodiments, modified nucleosides include 5-aza-cytidine,pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine,5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine,1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine,2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine,4-thio-1-methyl-pseudoisocytidine,4-thio-1-methyl-1-deaza-pseudoisocytidine,1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebulruine,5-methyl-zebularine, 5-aza-2-thio-zebulru.ine, 2-thio-zebulaiine,2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine,4-methoxy-pseudoisocytidine, and 4-methoxy-1-methyl-pseudoisocytidine.

In other embodiments, modified nucleosides include 2-aminopurine,2,6-diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine,7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine,7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine,1-methyladenosine, N6-methyladenosine, N6-isopentenyladenosine,N6-(cis-hydroxyisopentenyl)adenosine,2-methylthio-N6-(cis-hydroxyisopentenyl) adenosine,N6-glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine,2-methylthio-N6-threonyl carbamoyladenosine, N6,N6-dimethyladenosine,7-methyladenine, 2-methylthio-adenine, and 2-methoxy-adenine.

In specific embodiments, a modified nucleoside is5′-O-(1-Thiophosphate)-Adenosine, 5′-O-(1-Thiophosphate)-Cytidine,5′-O-(1-thiophosphate)-Guanosine, 5′-O-(1-Thiophophate)-Uridine or5′-O-(1-Thiophosphate)-Pseudouridine.

The α-thio substituted phosphate moiety is provided to confer stabilityto RNA and DNA polymers through the unnatural phosphorothioate backbonelinkages. Phosphorothioate DNA and RNA have increased nucleaseresistance and subsequently a longer half-life in a cellularenvironment. Phosphorothioate linked nucleic acids are expected to alsoreduce the innate immune response through weaker binding/activation ofcellular innate immune molecules.

In certain embodiments it is desirable to intracellularly degrade amodified nucleic acid introduced into the cell, for example if precisetiming of protein production is desired. Thus, the disclosure provides amodified nucleic acid containing a degradation domain, which is capableof being acted on in a directed manner within a cell.

In other embodiments, modified nucleosides include inosine,1-methyl-inosine, wyosine, wybutosine, 7-deaza-guanosine,7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine,6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine,6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine,1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine,8-oxo-guanosine, 7-methyl-8-oxo-guanosine, J-methyl-6-thio-guanosine,N2-methyl-6-thio-guanosine, and N2,N2-dimethyl-6-thio-guanosine.

Other components of nucleic acid are optional, and are beneficial insome embodiments. For example, a 5′ untranslated region (UTR) and/or a3′UTR are provided, wherein either or both may independently contain oneor more different nucleoside modifications. In such embodiments,nucleoside modifications may also be present in the translatable region.Also provided are nucleic acids containing a Kozak sequence.

Additionally, provided are nucleic acids containing one or more intronicnucleotide sequences capable of being excised from the nucleic acid.

Further, provided are nucleic acids containing an internal ribosomeentry site (IRES). An IRES may act as the sole ribosome binding site, ormay serve as one of multiplelibosome binding sites of an mRNA. An mRNAcontaining more than one functional ribosome binding site may encodeseveral peptides or polypeptides that are translated independently bythe ribosomes (“multicistronic mRNA”). When nucleic acids are providedwith an IRES, further optionally provided is a second translatableregion. Examples of IRES sequences that can be used according to thedisclosure include without limitation, those from picornaviruses (e.g.FMDV), pest viruses (CFFV), polio viruses (PV), encephalomyocarditisviruses (ECMV), foot-and-mouth disease viruses (FMDV), hepatitis Cviruses (HCV), classical swine fever viruses (CSFV), murine leukemiavirus (MLV), simian immune deficiency viruses (STY) or cricket paralysisviruses (CrPV).

Prevention or Reduction of Innate Cellular Immune Response ActivationUsing Modified Nucleic Acids.

The modified nucleic acids described herein are capable of evading aninnate immune response of a cell into which the nucleic acids areintroduced, thus increasing the efficiency of protein production in thecell. The term “innate immune response” includes a cellular response toexogenous single stranded nucleic acids, generally of viral or bacterialorigin, which involves the induction of cytokine expression and release,particularly the interferons, and cell death. Protein synthesis is alsoreduced during the innate cellular immune response. While it isadvantageous to eliminate the innate immune response in a cell, thedisclosure provides modified mRNAs that substantially reduce the immuneresponse, including Interferon signaling, without entirely eliminatingsuch a response. In some embodiments, the immune response is reduced byabout 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.9%, orgreater than 99.9%, as compared to the immune response induced by acorresponding unmodified nucleic acid. Such a reduction can be measuredby expression or activity level of Type 1 interferons or the expressionof interferon-regulated genes such as the toll-like receptors (e.g.,TLR7 and TLR8). Reduction of innate immune response can also be measuredby decreased cell death following one or more administrations ofmodified RNAs to a cell population; e.g., cell death is about 10%, 25%,50%, 75%, 85%, 90%, 95%, or over 95% less than the cell death frequencyobserved with a corresponding unmodified nucleic acid. Moreover, celldeath may affect fewer than about 50%, 40%, 30%, 20%, 10%, 5%, 1%, 0.1%,0.01%, or fewer than 0.01% of cells contacted with the modified nucleicacids.

The disclosure provides for the repeated introduction (e.g.,transfection) of modified nucleic acids into a target cell population,e.g., in vitro, ex vivo, or in vivo. The step of contacting the cellpopulation may be repeated one or more times (such as two, three, four,five, or more than five times). In some embodiments, the step ofcontacting the cell population with the modified nucleic acids isrepeated a number of times sufficient such that a predeterminedefficiency of protein translation in the cell population is achieved.Given the reduced cytotoxicity of the target cell population provided bythe nucleic acid modifications, such repeated transfections areachievable in a diverse array of cell types.

Modified Nucleic Acid Synthesis.

Nucleic acids for use in accordance with the disclosure may be preparedaccording to any available technique including, but not limited tochemical synthesis, enzymatic synthesis, which is generally termed invitro transcription, enzymatic or chemical cleavage of a longerprecursor, etc. Methods of synthesizing RNAs are known in the art (see,e.g., Gait, M. J. (ed.) Oligonucleotide synthesis: a practical approach,Oxford [Oxfordshire], Washington, D.C.: IRL Press, 1984; and Herdewijn,P. (ed.) Oligonucleotide synthesis: methods and applications, Methods inMolecular Biology, v. 288 (Clifton, N.J.) Totowa, N.J.: Humana Press,2005; both of which are incorporated herein by reference).

Modified nucleic acids need not be uniformly modified along the entirelength of the molecule. Different nucleotide modifications and/orbackbone structures may exist at various positions in the nucleic acid.One of ordinary skill in the alt will appreciate that the nucleotideanalogs or other modification(s) may be located at any position(s) of anucleic acid such that the function of the nucleic acid is notsubstantially decreased. A modification may also be a 5′ or 3′ terminalmodification. The nucleic acids may contain at a minimum one and atmaximum 100% modified nucleotides, or any intervening percentage, suchas at least about 50% modified nucleotides, at least about 80% modifiednucleotides, or at least about 90% modified nucleotides.

Generally, the length of a modified mRNA of the present disclosure issuitable for protein, polypeptide, or peptide production in a cell(e.g., a human cell). For example, the mRNA is of a length sufficient toallow translation of at least a dipeptide in a cell. In one embodiment,the length of the modified mRNA is greater than 30 nucleotides. Inanother embodiment, the length is greater than 35 nucleotides. Inanother embodiment, the length is at least 40 nucleotides. In anotherembodiment, the length is at least 45 nucleotides. In anotherembodiment, the length is at least 55 nucleotides. In anotherembodiment, the length is at least 60 nucleotides. In anotherembodiment, the length is at least 60 nucleotides. In anotherembodiment, the length is at least 80 nucleotides. In anotherembodiment, the length is at least 90 nucleotides. In anotherembodiment, the length is at least 100 nucleotides. In anotherembodiment, the length is at least 120 nucleotides. In anotherembodiment, the length is at least 140 nucleotides. In anotherembodiment, the length is at least 160 nucleotides. In anotherembodiment, the length is at least 180 nucleotides. In anotherembodiment, the length is at least 200 nucleotides. In anotherembodiment, the length is at least 250 nucleotides. In anotherembodiment, the length is at least 300 nucleotides. In anotherembodiment, the length is at least 350 nucleotides. In anotherembodiment, the length is at least 400 nucleotides. In anotherembodiment, the length is at least 450 nucleotides. In anotherembodiment, the length is at least 500 nucleotides. In anotherembodiment, the length is at least 600 nucleotides. In anotherembodiment, the length is at least 700 nucleotides. In anotherembodiment, the length is at least 800 nucleotides. In anotherembodiment, the length is at least 900 nucleotides. In anotherembodiment, the length is at least 1000 nucleotides. In anotherembodiment, the length is at least 1100 nucleotides. In anotherembodiment, the length is at least 1200 nucleotides. In anotherembodiment, the length is at least 1300 nucleotides. In anotherembodiment, the length is at least 1400 nucleotides. In anotherembodiment, the length is at least 1500 nucleotides. In anotherembodiment, the length is at least 1600 nucleotides. In anotherembodiment, the length is at least 1800 nucleotides. In anotherembodiment, the length is at least 2000 nucleotides. In anotherembodiment, the length is at least 2500 nucleotides. In anotherembodiment, the length is at least 3000 nucleotides. In anotherembodiment, the length is at least 4000 nucleotides. In anotherembodiment, the length is at least 5000 nucleotides, or greater than5000 nucleotides.

Uses of Modified Nucleic Acids.

The proteins, polypeptides, or peptides produced by the methodsdescribed herein can be used as therapeutic agents to treat or preventone or more diseases or conditions described herein.

Therapeutic Agents.

Provided are compositions, methods, kits, and reagents for treatment orprevention of disease or conditions in humans and other animals (e.g.,mammals). The active therapeutic agents of the disclosure includepolypeptides translated from modified nucleic acids, cells containingmodified nucleic acids or polypeptides translated from the modifiednucleic acids, and cells contacted with cells containing modifiednucleic acids or polypeptides translated from the modified nucleicacids.

Provided are methods of inducing translation of a recombinantpolypeptide in a cell population using the modified nucleic acidsdescribed herein. Such translation can be in vivo, ex vivo, in culture,or in vitro. The cell population is contacted with an effective amountof a composition containing a nucleic acid that has at least onenucleoside modification, and a translatable region encoding therecombinant polypeptide. The population is contacted under conditionssuch that the nucleic acid is localized into one or more cells of thecell population and the recombinant polypeptide is translated in thecell from the nucleic acid.

An effective amount of the composition is provided based, at least inpart, on the target tissue, target cell type, means of administration,physical characteristics of the protein translated from the modifiednucleic acid (e.g., size), and other determinants.

Compositions containing modified nucleic acids are formulated foradministration intramuscularly, transarterially, intraperitoneally,intravenously, intranasally, subcutaneously, endoscopically,transdermally, or intrathecally. In some embodiments, the composition isformulated for extended release.

The subject to whom the therapeutic agent is administered suffers fromor is at risk of developing a disease, disorder, or deleteriouscondition. Provided are methods of identifying, diagnosing, andclassifying subjects on these bases, which may include clinicaldiagnosis, biomarker levels, genome-wide association studies (GWAS), andother methods known in the art.

In certain embodiments, the administered recombinant polypeptidetranslated from the modified nucleic acid described herein provide afunctional activity which is substantially absent in the cell in whichthe recombinant polypeptide is administered. For example, the missingfunctional activity may be enzymatic, structural, or gene regulatory innature.

In other embodiments, the administered recombinant polypeptide replacesa polypeptide (or multiple polypeptides) that is substantially absent inthe cell in which the recombinant polypeptide is administered. Suchabsence may be due to genetic mutation of the encoding gene orregulatory pathway thereof. Alternatively, the recombinant polypeptidefunctions to antagonize the activity of an endogenous protein presentin, on the surface of, or secreted from the cell. Usually, the activityof the endogenous protein is deleterious to the subject, for example,due to mutation of the endogenous protein resulting in altered activityor localization. Additionally, the recombinant polypeptide antagonizes,directly or indirectly, the activity of a biological moiety present in,on the surface of, or secreted from the cell. Examples of antagonizedbiological moieties include lipids (e.g., cholesterol), a lipoprotein(e.g., low density lipoprotein), a nucleic acid, a carbohydrate, or asmall molecule toxin.

The recombinant proteins described herein are engineered forlocalization within the cell, potentially within a specific compartmentsuch as the nucleus, or are engineered for secretion from the cell ortranslocation to the plasma membrane of the cell.

As described herein, a useful feature of the modified nucleic acids ofthe disclosure is the capacity to reduce the innate immune response of acell to an exogenous nucleic acid, e.g., to increase protein production.Provided are methods for performing the titration, reduction orelimination of the immune response in a cell or a population of cells.In some embodiments, the cell is contacted with a first composition thatcontains a first dose of a first exogenous nucleic acid including atranslatable region and at least one nucleoside modification, and thelevel of the innate immune response of the cell to the first exogenousnucleic acid is determined. Subsequently, the cell is contacted with asecond composition, which includes a second dose of the first exogenousnucleic acid, the second dose containing a lesser amount of the firstexogenous nucleic acid as compared to the first dose. Alternatively, thecell is contacted with a first dose of a second exogenous nucleic acid.The second exogenous nucleic acid may contain one or more modifiednucleosides, which may be the same or different from the first exogenousnucleic acid or, alternatively, the second exogenous nucleic acid maynot contain modified nucleosides. The steps of contacting the cell withthe first composition and/or the second composition may be repeated oneor more times. Additionally, efficiency of protein production

(e.g., protein translation) in the cell is optionally determined, andthe cell may be re-transfected with the first and/or second compositionrepeatedly until a target protein production efficiency is achieved.

Therapeutics for Diseases and Conditions.

Provided are methods for treating or preventing a symptom of diseasescharacterized by missing or aberrant protein activity, by replacing themissing protein activity or overcoming the aberrant protein activity.

Diseases characterized by dysfunctional or aberrant protein activityinclude, but not limited to, cancer and proliferative diseases, geneticdiseases (e.g., cystic fibrosis), autoimmune diseases, diabetes,neurodegenerative diseases, cardiovascular diseases, and metabolicdiseases. The present disclosure provides a method for treating suchconditions or diseases in a subject by introducing protein or cell-basedtherapeutics produced by a method using the modified nucleic acidsprovided herein, wherein the modified nucleic acids encode for a proteinthat antagonizes or otherwise overcomes the aberrant protein activitypresent in the cell of the subject. Specific examples of a dysfunctionalprotein are the missense mutation variants of the cystic fibrosistransmembrane conductance regulator (CFTR) gene, which produce adysfunctional protein variant of CFTR protein, which causes cysticfibrosis.

Multiple diseases are characterized by missing (or substantiallydiminished such that proper protein function does not occur) proteinactivity. Such proteins may not be present, or are essentiallynon-functional. The present disclosure provides a method for treatingsuch conditions or diseases in a subject by introducing nucleic acid orcell-based therapeutics containing the modified nucleic acids providedherein, wherein the modified nucleic acids encode for a protein thatreplaces the protein activity missing from the target cells of thesubject. Specific examples of a dysfunctional protein are the nonsensemutation variants of the cystic fibrosis transmembrane conductanceregulator (CFTR) gene, which produce a nonfunctional protein valiant ofCFTR protein, which causes cystic fibrosis.

Thus, provided are methods of treating cystic fibrosis in a mammaliansubject by contacting a cell of the subject with a modified nucleic acidhaving a translatable region that encodes a functional CFTR polypeptide,under conditions such that an effective amount of the CTFR polypeptideis present in the cell. Typical target cells are epithelial cells, suchas the lung, and methods of administration are determined in view of thetarget tissue; i.e., for lung delivery, the RNA molecules are formulatedfor administration by inhalation.

In another embodiment, the present disclosure provides a method fortreating hyperlipidemia in a subject, by introducing into a cellpopulation of the subject with Sortilin (a protein recentlycharacterized by genomic studies) produced by a method described hereinusing a modified mRNA molecule encoding Sortilin, thereby amelioratingthe hyperlipidemia in a subject. The SORT1 gene encodes a trans-Golginetwork (TGN) transmembrane protein called Sortilin. Genetic studieshave shown that one of five individuals has a single nucleotidepolymorphism, rs12740374, in the 1p13 locus of the SORT1 gene thatpredisposes them to having low levels of low-density lipoprotein (LDL)and very-low-density lipoprotein (VLDL). Each copy of the minor allele,present in about 30% of people, alters LDL cholesterol by 8 mg/dL, whiletwo copies of the minor allele, present in about 5% of the population,lowers LDL cholesterol 16 mg/dL. Carriers of the minor allele have alsobeen shown to have a 40% decreased risk of myocardial infarction.Functional in vivo studies in mice describes that

overexpression of SORT1 in mouse liver tissue led to significantly lowerLDL-cholesterol levels, as much as 80% lower, and that silencing SORT1increased LDL cholesterol approximately 200% (Musunuru K. et al. Fromnoncoding variant to phenotype via SORT1 at the 1p13 cholesterol locus.Nature 2010; 466: 714-721).

Pharmaceutical Compositions

The present disclosure provides proteins generated from modified mRNAsand proteins produced by the methods described herein can be used inpharmaceutical compositions. Pharmaceutical compositions may optionallycomprise one or more additional therapeutically active substances. Inaccordance with some embodiments, a method of administeringpharmaceutical compositions comprising one or more proteins to bedelivered to a subject in need thereof is provided. In some embodiments,compositions are administered to humans. For the purposes of the presentdisclosure, the phrase “active ingredient” generally refers to a proteinor protein-containing complex as described herein.

Although the descriptions of pharmaceutical compositions provided hereinare principally directed to pharmaceutical compositions which aresuitable for administration to humans, it will be understood by theskilled artisan that such compositions are generally suitable foradministration to animals of all sorts. Modification of pharmaceuticalcompositions suitable for administration to humans in order to renderthe compositions suitable for administration to various animals is wellunderstood, and the ordinarily skilled veterinary pharmacologist candesign and/or perform such modification with merely ordinary, if any,experimentation. Subjects to which administration of the pharmaceuticalcompositions is contemplated include, but are not limited to, humansand/or other primates; mammals, including commercially relevant mammalssuch as cattle, pigs, horses, sheep, cats, dogs, mice, and/or rats;and/or birds, including commercially relevant birds such as chickens,ducks, geese, and/or turkeys.

Formulations of the pharmaceutical compositions described herein may beprepared by any method known or hereafter developed in the art ofpharmacology. In general, such preparatory methods include the step ofbringing the active ingredient into association with an excipient and/orone or more other accessory ingredients, and then, if necessary and/ordesirable, shaping and/or packaging the product into a desired single-or multi-dose unit.

A pharmaceutical composition in accordance with the disclosure may beprepared, packaged, and/or sold in bulk, as a single unit dose, and/oras a plurality of single unit doses. As used herein, a “unit dose” isdiscrete amount of the pharmaceutical composition comprising apredetermined amount of the active ingredient. The amount of the activeingredient is generally equal to the dosage of the active ingredientwhich would be administered to a subject and/or a convenient fraction ofsuch a dosage such as, for example, one-half or one-third of such adosage.

Relative amounts of the active ingredient, the pharmaceuticallyacceptable excipient, and/or any additional ingredients in apharmaceutical composition in accordance with the disclosure will vary,depending upon the identity, size, and/or condition of the subjectheated and further depending upon the route by which the composition isto be administered. By way of example, the composition may comprisebetween 0.1% and 100% (w/w) active ingredient.

Pharmaceutical compositions may be formulated to additionally comprise apharmaceutically acceptable excipient, which, as used herein, includesany and all solvents, dispersion media, diluents, or other liquidvehicles, dispersion or suspension aids, surface active agents, isotonicagents, thickening or emulsifying agents, preservatives, solid binders,lubricants and the like, as suited to the particular dosage formdesired. Remington's The Science and Practice of Pharmacy, 21₅₁ Edition,A. R. Gennaro (Lippincott, Williams & Wilkins, Baltimore, Md., 2006;incorporated herein by reference) discloses various excipients used informulating pharmaceutical compositions and known techniques for thepreparation thereof. Except insofar as any conventional excipient mediumis incompatible with a substance or its derivatives, such as byproducing any undesirable biological effect or otherwise interacting ina deleterious manner with any other component(s) of the pharmaceuticalcomposition, its use is contemplated to be within the scope of thisdisclosure.

In some embodiments, a pharmaceutically acceptable excipient is at least95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%pure. In some embodiments, an excipient is approved for use in humansand for veterinary use. In some embodiments, an excipient is approved byUnited States Food and Drug Administration. In some embodiments,

an excipient is pharmaceutical grade. In some embodiments, an excipientmeets the standards of the United States Pharmacopoeia (USP), theEuropean Pharmacopoeia (EP), the British Pharmacopoeia, and/or theInternational Pharmacopoeia.

Pharmaceutically acceptable excipients used in the manufacture ofpharmaceutical compositions include, but are not limited to, inertdiluents, dispersing and/or granulating agents, surface active agentsand/or emulsifiers, disintegrating agents, binding agents,preservatives, buffering agents, lubricating agents, and/or oils. Suchexcipients may optionally be included in pharmaceutical compositions.Excipients such as cocoa butter and suppository waxes, coloring agents,coating agents, sweetening, flavoring, and/or perfuming agents can bepresent in the composition, according to the judgment of the formulator.

Exemplary diluents include, but are not limited to, calcium carbonate,sodium carbonate, calcium phosphate, dicalcium phosphate, calciumsulfate, calcium hydrogen phosphate, sodium phosphate lactose, sucrose,cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol,inositol, sodium chloride, dry starch, cornstarch, powdered sugar, etc.,and/or combinations thereof.

Exemplary granulating and/or dispersing agents include, but are notlimited to, potato starch, corn starch, tapioca starch, sodium starchglycolate, clays, alginic acid, guar gum, citrus pulp, agar, bentonite,cellulose and wood products, natural sponge, cation-exchange resins,calcium carbonate, silicates, sodium carbonate, cross-linkedpoly(vinyl-pyrrolidone) (crospovidone), sodium carboxymethyl starch(sodium starch glycolate), carboxymethyl cellulose, cross-linked sodiumcarboxymethyl cellulose (croscarmellose), methylcellulose,pregelatinized starch (starch 1500), microcrystalline starch, waterinsoluble starch, calcium carboxymethyl cellulose, magnesium aluminumsilicate (Veegum), sodium lauryl sulfate, quaternary ammonium compounds,etc., and/or combinations thereof.

Exemplary surface active agents and/or emulsifiers include, but are notlimited to, natural emulsifiers (e.g., acacia, agar, alginic acid,sodium alginate, tragacanth, chondrux, cholesterol, xanthan, pectin,gelatin, egg yolk, casein, wool fat, cholesterol, wax, and lecithin),colloidal clays (e.g., bentonite [aluminum silicate] and Veegum®[magnesium aluminum silicate]), long chain amino acid derivatives, highmolecular weight alcohols (e.g., stearyl alcohol, cetyl alcohol, oleylalcohol, triacetin monostearate, ethylene glycol distearate, glycerylmonostearate, and propylene glycol monostearate, polyvinyl alcohol),carbomers (e.g. carboxy polymethylene, polyacrylic acid, acrylic acidpolymer, and carboxyvinyl polymer), carrageenan, cellulosic derivatives(e.g., carboxymethylcellulose sodium, powdered cellulose, hydroxymethylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose,methylcellulose), sorbitan fatty acid esters (e.g., polyoxyethylenesorbitan monolaurate [Tween®20], polyoxyethylene sorbitan [Tween®60],polyoxyethylene sorbitan monoleate [Tween®80], sorbitan monopalmitate[Span®40], sorbitan monostearate [Span®60], sorbitan tristearate[Span®65], glyceryl monoleate, sorbitan monoleate [Span®80]),polyoxyethylene esters (e.g., polyoxyethylene monostearate [Myrj®45],polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil,polyoxymethylene stearate, and Solutol®), sucrose fatty acid esters,polyethylene glycol fatty acid esters (e.g., Cremophor®),polyoxyethylene ethers, (e.g., polyoxyethylene lauryl ether [Brij®30]),poly(vinyl-pyrrolidone), diethylene glycol monolaurate, triethanolamineoleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyllaurate, sodium lauryl sulfate, Pluronic®F 68, Poloxamer®188,cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride,docusate sodium, etc. and/or combinations thereof.

Exemplary binding agents include, but are not limited to, starch (e.g.,cornstarch and starch paste); gelatin; sugars (e.g., sucrose, glucose,dextrose, dextrin, molasses, lactose, lactitol, mannitol,); natural andsynthetic gums (e.g., acacia, sodium alginate, extract of Irish moss,panwar gum, ghatti gum, mucilage of isapol husks,carboxymethylcellulose, methylcellulose, ethylcellulose,hydroxyethylcellulose, hydroxypropyl cellulose, hydroxypropylmethylcellulose, microcrystalline cellulose, cellulose acetate,poly(vinyl-pyrrolidone), magnesium aluminum silicate (Veegum®), andlarch arabogalactan); alginates; polyethylene oxide; polyethyleneglycol; inorganic calcium salts; silicic acid; polymethacrylates; waxes;water; alcohol; etc.; and combinations thereof.

Exemplary preservatives may include, but are not limited to,antioxidants, chelating agents, antimicrobial preservatives, antifungalpreservatives, alcohol preservatives, acidic preservatives, and/or otherpreservatives. Exemplary antioxidants include, but are not limited to,alpha tocopherol, ascorbic acid, acorbyl palmitate, butylatedhydroxyanisole, butylated hydroxytoluene, monothioglycerol, potassiummetabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodiumbisulfite, sodium metabisulfite, and/or sodium sulfite. Exemplarychelating agents include ethylenediaminetetraacetic acid (EDTA), citricacid monohydrate, disodium edetate, dipotassium edetate, edetic acid,fumaric acid, malic acid, phosphoric acid, sodium edetate, trulalicacid, and/or trisodium edetate. Exemplary antimicrobial preservativesinclude, but are not limited to, benzalkonium chloride, benzethoniumchloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride,chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethylalcohol, glycerin, hexetidine, imidurea, phenol, phenoxyethanol,phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, and/orthimerosal. Exemplary antifungal preservatives include, but are notlimited to, butyl paraben, methyl paraben, ethyl paraben, propylparaben, benzoic acid, hydroxybenzoic acid, potassium benzoate,potassium sorbate, sodium benzoate, sodium propionate, and/or sorbicacid. Exemplary alcohol preservatives include, but are not limited to,ethanol, polyethylene glycol, phenol, phenolic compounds, bisphenol,chlorobutanol, hydroxybenzoate, and/or phenylethyl alcohol. Exemplaryacidic preservatives include, but are not limited to, vitamin A, vitaminC, vitamin E, beta-carotene, citric acid, acetic acid, dehydroaceticacid, ascorbic acid, sorbic acid, and/or phytic

acid. Other preservatives include, but are not limited to, tocopherol,tocopherol acetate, deteroxime mesylate, cetrimide, butylatedhydroxyanisol (BHA), butylated hydroxytoluened (BHT), ethylenediamine,sodium lauryl sulfate (SLS), sodium lauryl ether sulfate (SLES), sodiumbisulfite, sodium metabisulfite, potassium sulfite, potassiummetabisulfite, Glydant Plus®, Phenonip®, methylparaben, Germall®115,Germaben®II, Neolone™, Kathon™ and/or Euxyl®.

Exemplary buffering agents include, but are not limited to, citratebuffer solutions, acetate buffer solutions, phosphate buffer solutions,ammonium chloride, calcium carbonate, calcium chloride, calcium citrate,calcium glubionate, calcium gluceptate, calcium gluconate, o-gluconicacid, calcium glycerophosphate, calcium lactate, propanoic acid, calciumlevulinate, pentanoic acid, dibasic calcium phosphate, phosphoric acid,tribasic calcium phosphate, calcium hydroxide phosphate, potassiumacetate, potassium chloride, potassium gluconate, potassium mixtures,dibasic potassium phosphate, monobasic potassium phosphate, potassiumphosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride,sodium citrate, sodium lactate, dibasic sodium phosphate, monobasicsodium phosphate, sodium phosphate mixtures, tromethamine, magnesiumhydroxide, aluminum hydroxide, alginic acid, pyrogen-free water,isotonic saline, Ringer's solution, ethyl alcohol, etc., and/orcombinations thereof.

Exemplary lubricating agents include, but are not limited to, magnesiumstearate, calcium stearate, stearic acid, silica, talc, malt, glycerylbehanate, hydrogenated vegetable oils, polyethylene glycol, sodiumbenzoate, sodium acetate, sodium chloride, leucine, magnesium laurylsulfate, sodium lauryl sulfate, etc., and combinations thereof.

Exemplary oils include, but are not limited to, almond, apricot kernel,avocado, babassu, bergamot, black current seed, borage, cade, camomile,canola, caraway, carnauba, castor, cinnamon, cocoa butter, coconut, codliver, coffee, corn, cotton seed, emu, eucalyptus, evening primrose,fish, flaxseed, geraniol, gourd, grape seed, hazel nut, hyssop,isopropyl mylistate, jojoba, kukui nut, lavandin, lavender, lemon,litsea cubeba, macademia nut, mallow, mango seed, meadowfoam seed, mink,nutmeg, olive, orange, orange roughy, palm, palm kernel, peach kernel,peanut, poppy seed, pumpkin seed, rapeseed, rice bran, rosemary,safflower, sandalwood, sasquana, savoury, sea buckthorn, sesame, sheabutter, silicone, soybean, sunflower, tea tree, thistle, tsubaki,vetiver, walnut, and wheat germ oils. Exemplary oils include, but arenot limited to, butyl stearate, caprylic triglyceride, caprictriglyceride, cyclomethicone, diethyl sebacate, dimethicone 360,isopropyl myristate, mineral oil, octyldodecanol, oleyl alcohol,silicone oil, and/or combinations thereof.

Liquid dosage forms for oral and parenteral administration include, butare not limited to, pharmaceutically acceptable emulsions,microemulsions, solutions, suspensions, syrups, and/or elixirs. Inaddition to active ingredients, liquid dosage forms may comprise inertdiluents commonly used in the art such as, for example, water or othersolvents, solubilizing agents and emulsifiers such as ethyl alcohol,isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol,benzyl benzoate, propylene glycol, 1,3-butylene glycol,dimethylformamide, oils (in particular, cottonseed, groundnut, corn,germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfurylalcohol, polyethylene glycols and fatty acid esters of sorbitan, andmixtures thereof. Besides inert diluents, oral compositions can includeadjuvants such as wetting agents, emulsifying and suspending agents,sweetening, flavoring, and/or perfuming agents. In certain embodimentsfor parenteral administration, compositions are mixed with solubilizingagents such as Cremophor®, alcohols, oils, modified oils, glycols,polysorbates, cyclodextrins, polymers, and/or combinations thereof.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions may be formulated according to the known artusing suitable dispersing agents, wetting agents, and/or suspendingagents. Sterile injectable preparations may be sterile injectablesolutions, suspensions, and/or emulsions in nontoxic parenterallyacceptable diluents and/or solvents, for example, as a solution in1,3-butanediol. Among the acceptable vehicles and solvents that may beemployed are water, Ringer's solution, U.S.P., and isotonic sodiumchloride solution. Sterile, fixed oils are conventionally employed as asolvent or suspending medium. For this purpose any bland fixed oil canbe employed including synthetic mono- or diglycerides. Fatty acids suchas oleic acid can be used in the preparation of injectables.

Injectable compositions can be sterilized, for example, by filtrationthrough a bacterial-retaining filter, and/or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile injectablemedium prior to use.

In order to prolong the effect of an active ingredient, it is oftendesirable to slow the absorption of the active ingredient fromsubcutaneous or intramuscular injection. This may be accomplished by theuse of a liquid suspension of crystalline or amorphous material withpoor water solubility. The rate of absorption of the drug then dependsupon its rate of dissolution which, in turn, may depend upon crystalsize and crystalline form. Alternatively, delayed absorption of aparenterally administered drug form is accomplished by dissolving orsuspending the drug in an oil vehicle. Injectable depot forms are madeby forming microencapsule matrices of the drug in biodegradable polymerssuch as polylactide-polyglycolide. Depending upon the ratio of drug topolymer and the nature of the particular polymer employed, the rate ofdrug release can be controlled. Examples of other biodegradable polymersinclude poly(orthoesters) and poly(anhydrides). Depot injectablecompositions are formulated or prepared by entrapping the drug inliposomes or microemulsions which are compatible with body tissues.

Compositions for rectal or vaginal administration are typicallysuppositories which can be prepared by mixing compositions with suitablenon-irritating excipients such as cocoa butter, polyethylene glycol or asuppository wax which are solid at ambient temperature but liquid atbody temperature and therefore melt in the rectum or vaginal cavity andrelease the active ingredient.

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, an activeingredient is mixed with at least one inert, pharmaceutically acceptableexcipient such as sodium citrate or dicalcium phosphate and/or fillersor extenders (e.g., starches, lactose, sucrose, glucose, mannitol, andsilicic acid), binders (e.g., carboxymethylcellulose, alginates,gelatin, polyvinylpyrrolidinone, sucrose, and acacia), humectants (e.g.glycerol), disintegrating agents (e.g., agar, calcium carbonate, potatoor tapioca starch, alginic acid, certain silicates, and sodiumcarbonate), solution retarding agents (e.g., paraffin), absorptionaccelerators (e.g., quaternary ammonium compounds), wetting agents(e.g., cetyl alcohol and glycerol monostearate), absorbents (e.g.,kaolin and bentonite clay), and lubricants (e.g., talc, calciumstearate, magnesium stearate, solid polyethylene glycols, sodium laurylsulfate), and mixtures thereof. In the case of capsules, tablets andpills, the dosage form may comprise buffering agents.

Solid compositions of a similar type may be employed as fillers in softand hard-filled gelatin capsules using such excipients as lactose ormilk sugar as well as high molecular weight polyethylene glycols and thelike. Solid dosage forms of tablets, dragees, capsules, pills, andgranules can be prepared with coatings and shells such as entericcoatings and other coatings well known in the pharmaceutical formulatingrut. They may optionally comprise opacifying agents and can be of acomposition that they release the active ingredient(s) only, orpreferentially, in a certain part of the intestinal tract, optionally,in a delayed manner. Examples of embedding compositions which can beused include polymeric substances and waxes. Solid

compositions of a similar type may be employed as fillers in soft andhard-filled gelatin capsules using such excipients as lactose or milksugar as well as high molecular weight polyethylene glycols and thelike.

Dosage forms for topical and/or transdermal administration of acomposition may include ointments, pastes, creams, lotions, gels,powders, solutions, sprays, inhalants and/or patches. Generally, anactive ingredient is admixed under sterile conditions with apharmaceutically acceptable excipient and/or any needed preservativesand/or buffers as may be required. Additionally, the present disclosurecontemplates the use of transdermal patches, which often have the addedadvantage of providing controlled delivery of a compound to the body.Such dosage forms may be prepared, for example, by dissolving and/ordispensing the compound in the proper medium. Alternatively oradditionally, rate may be controlled by either providing a ratecontrolling membrane and/or by dispersing the compound in a polymermatrix ruld/or gel.

Suitable devices for use in delivering intradermal pharmaceuticalcompositions described herein include short needle devices such as thosedescribed in U.S. Pat. Nos. 4,886,499; 5,190,521; 5,328,483; 5,527,288;4,270,537; 5,015,235; 5,141,496; and 5,417,662. Intradermal compositionsmay be administered by devices which limit the effective penetrationlength of a needle into the skin, such as those described in PCTpublication WO 99/34850 and functional equivalents thereof. Jetinjection devices which deliver liquid compositions to the dermis via aliquid jet injector and/or via a needle which pierces the stratumcorneum and produces a jet which reaches the dermis are suitable. Jetinjection devices are described, for example, in U.S. Pat. Nos.5,480,381; 5,599,302; 5,334,144; 5,993,412; 5,649,912; 5,569,189;5,704,911; 5,383,851; 5,893,397; 5,466,220; 5,339,163; 5,312,335;5,503,627; 5,064,413; 5,520,639; 4,596,556; 4,790,824; 4,941,880;4,940,460; and PCT publications WO 97/37705 and WO 97/13537. Ballisticpowder/particle delivery devices which use compressed gas to acceleratevaccine in powder form through the outer layers of the skin to thedermis are suitable. Alternatively or additionally, conventionalsyringes may be used in the classical mantoux method of intradermaladministration.

Compositions formulated for topical administration include, but are notlimited to, liquid and/or semi liquid preparations such as liniments,lotions, oil in water and/or water in oil emulsions such as creams,ointments and/or pastes, and/or solutions and/or suspensions.Topically-administrable compositions may be formulated, for example, tocomprise from about 1% to about 10% (w/w) active ingredient, althoughthe concentration of active ingredient may be as high as the solubilitylimit of the active ingredient in the solvent. Compositions formulatedfor topical administration may further comprise one or more of theadditional ingredients described herein.

A pharmaceutical composition may be formulated, prepared, packaged,and/or sold for pulmonary administration via the buccal cavity. Such acomposition may be formulated to comprise dry particles which comprisethe active ingredient and which have a diameter in the range from about0.5 nm to about 7 nm or from about 1 nm to about 6 nm. Such compositionsare conveniently in the form of dry powders for administration using adevice comprising a dry powder reservoir to which a stream of propellantmay be directed to disperse the powder and/or using a self propellingsolvent/powder dispensing container such as a device comprising theactive ingredient dissolved and/or suspended in a low-boiling propellantin a sealed container. Such powders comprise particles wherein at least98% of the particles by weight have a diameter greater than 0.5 nm andat least 95% of the particles by number have a diameter less than 7 nm.Alternatively, at least 95% of the particles by weight have a diametergreater than 1 nm and at least 90% of the particles by number have adiameter less than 6 nm. Dry powder compositions may include a solidfine powder diluent such as sugar and are conveniently provided in aunit dose form.

Low boiling propellants generally include liquid propellants having aboiling point of below 65° F. at atmospheric pressure. Generally thepropellant may constitute about 50% to about 99.9% (w/w) of thecomposition, and active ingredient may constitute about 0.1% to about20% (w/w) of the composition. A propellant may further compriseadditional ingredients such as a liquid non-ionic and/or solid anionicsurfactant and/or a solid diluent (which may have a particle size of thesame order as particles comprising the active ingredient).

Pharmaceutical compositions formulated for pulmonary delivery mayprovide an active ingredient in the form of droplets of a solutionand/or suspension. Such compositions may be formulated, prepared,packaged, and/or sold as aqueous and/or dilute alcoholic solutionsand/or suspensions, optionally sterile, comprising active ingredient,and may conveniently be administered using an y nebulization and/oratomization device. Such compositions may further comprise one or moreadditional ingredients including, but not limited to, a flavoring agentsuch as saccharin sodium, a volatile oil, a buffering agent, a surfaceactive agent, and/or a preservative such as methylhydroxybenzoate.Droplets provided by this route of administration may have an averagediameter in the range from about 0.1 nm to about 200 nm.

Formulations described herein as being useful for pulmonary deliveryrule useful for intranasal delivery of a pharmaceutical composition.Another composition formulated for intranasal administration is a coarsepowder comprising the active ingredient and having an average particlefrom about 0.2 μm to 500 μm. Such a composition is formulated foradministration in the manner in which snuff is taken, i.e. by rapidinhalation through the nasal passage from a container of the powder heldclose to the nose.

Compositions formulated for nasal administration may, for example,comprise from about as little as 0.1% (w/w) and as much as 100% (w/w) ofactive ingredient, and may comprise one or more of the additionalingredients described herein. A pharmaceutical composition may beformulated, prepared, packaged, and/or sold for buccal administration.Such compositions may, for example, be formulated in the form of tabletsand/or lozenges made using conventional methods, and may contain, forexample, 0.1% to 20% (w/w) active ingredient, the balance comprising anorally dissolvable and/or degradable composition and, optionally, one ormore of the additional ingredients described herein. Alternately,compositions formulated for buccal administration may comprise a powderand/or an aerosolized and/or atomized solution and/or suspensioncomprising active ingredient. Such powdered, aerosolized, and/oraerosolized compositions, when dispersed, may have an average particleand/or droplet size in the range from about 0.1 nm to about 200 nm, andmay further comprise one or more of any additional ingredients describedherein.

A pharmaceutical composition may be formulated, prepared, packaged,and/or sold for ophthalmic administration. Such compositions may, forexample, be formulated in the form of eye drops including, for example,a 0.1/1.0% (w/w) solution and/or suspension of the active ingredient inan aqueous or oily liquid excipient. Such drops may further comprisebuffering agents, salts, and/or one or more other of any additionalingredients described herein. Other opthalmically-administrablecompositions which are useful include those which comprise the activeingredient in microcrystalline form and/or in a liposomal preparation.Ear drops and/or eye drops are contemplated as being within the scope ofthis disclosure.

General considerations in the formulation and/or manufacture ofpharmaceutical agents may be found, for example, in Remington: TheScience and Practice of Pharmacy 21st ed., Lippincott Williams &Wilkins, 2005 (incorporated herein by reference).

Administration.

The present disclosure provides methods comprising administeringproteins or compositions produced by the methods described herein to asubject in need thereof. Proteins or complexes, or pharmaceutical,imaging, diagnostic, or prophylactic compositions thereof, may beadministered to a subject using any amount and any route ofadministration effective for preventing, treating, diagnosing, orimaging a disease, disorder, and/or condition (e.g., a disease,disorder, and/or condition relating to working memory deficits). Theexact amount required will vary from subject to subject, depending onthe species, age, and general condition of the subject, the severity ofthe disease, the particular composition, its mode of administration, itsmode of activity, and the like. Compositions in accordance with thedisclosure are typically formulated in dosage unit from for ease ofadministration and uniformity of dosage. It will be understood, however,that the total daily usage of the compositions of the present disclosurewill be decided by the attending physician within the scope of soundmedical judgment. The specific therapeutically effective,prophylactically effective, or appropriate imaging dose level for anyparticular patient will depend upon a variety of factors including thedisorder being treated and the severity of the disorder; the activity ofthe specific compound employed; the specific composition employed; theage, body weight, general health, sex and diet of the patient; the timeof administration, route of administration, and rate of excretion of thespecific compound employed; the duration of the treatment; drugs used incombination or coincidental with the specific compound employed; andlike factors well known in the medical arts.

Proteins to be delivered and/or pharmaceutical, prophylactic,diagnostic, or imaging compositions thereof may be administered toanimals, such as mammals (e.g., humans, domesticated animals, cats,dogs, mice, rats, etc.). In some embodiments, pharmaceutical,prophylactic, diagnostic, or imaging compositions thereof areadministered to humans.

Proteins to be delivered and/or pharmaceutical, prophylactic,diagnostic, or imaging compositions thereof in accordance with thepresent disclosure may be administered by any route. In someembodiments, proteins and/or pharmaceutical, prophylactic, diagnostic,or imaging compositions thereof, are administered by one or more of avariety of routes, including oral, intravenous, intramuscular,intra-artetial, intramedullary, intrathecal, subcutaneous,intraventricular, transdermal, interdermal, rectal, intravaginal,intraperitoneal, topical (e.g., by powders, ointments, creams, gels,lotions, and/or drops), mucosal, nasal, buccal, enteral, vitreal,intratumoral, sublingual; by intratracheal instillation, bronchialinstillation, and/or inhalation; as an oral spray, nasal spray, and/oraerosol, and/or through a portal vein catheter. In some embodiments,proteins or complexes, and/or pharmaceutical, prophylactic, diagnostic,or imaging compositions thereof, are administered by systemicintravenous injection. In specific embodiments, proteins or complexesand/or pharmaceutical, prophylactic, diagnostic, or imaging compositionsthereof may be administered intravenously and/or orally. In specificembodiments, proteins or complexes, and/or pharmaceutical, prophylactic,diagnostic, or imaging compositions thereof, may be administered in away which allows the protein or complex to cross the blood-brainbarrier, vascular barrier, or other epithelial barrier.

However, the disclosure encompasses the delivery of proteins orcomplexes, and/or pharmaceutical, prophylactic, diagnostic, or imagingcompositions thereof, by any appropriate route taking into considerationlikely advances in the sciences of drug delivery.

In general the most appropriate route of administration will depend upona variety of factors including the nature of the protein or complexcomprising proteins associated with at least one agent to be delivered(e.g., its stability in the environment of the gastrointestinal tract,bloodstream, etc.), the condition of the patient (e.g., whether thepatient is able to tolerate particular routes of administration), etc.The disclosure encompasses the delivery of the pharmaceutical,prophylactic, diagnostic, or imaging compositions by any appropriateroute taking into consideration likely advances in the sciences of drugdelivery.

In certain embodiments, compositions in accordance with the disclosuremay be administered at dosage levels sufficient to deliver from about0.0001 mg/kg to about 100 mg/kg, from about 0.01 mg/kg to about 50mg/kg, from about 0.1 mg/kg to about 40 mg/kg, from about 0.5 mg/kg toabout 30 mg/kg, from about 0.01 mg/kg to about 10 mg/kg, from about 0.1mg/kg to about 10 mg/kg, or from about 1 mg/kg to about 25 mg/kg, ofsubject body weight per day, one or more times a day, to obtain thedesired therapeutic, diagnostic, prophylactic, or imaging effect. Thedesired dosage may be delivered three times a day, two times a day, oncea day, every other day, every third day, every week, every two weeks,every three weeks, or every four weeks. In certain embodiments, thedesired dosage may be delivered using multiple administrations (e.g.,two, three, four, five, six, seven, eight, nine, ten, eleven, twelve,thirteen, fourteen, or more administrations).

Proteins or complexes may be used in combination with one or more othertherapeutic, prophylactic, diagnostic, or imaging agents. By “incombination with,” it is not intended to imply that the agents must beadministered at the same time and/or formulated for delivery together,although these methods of delivery are within the scope of thedisclosure. Compositions can be administered concurrently with, priorto, or subsequent to, one or more other desired therapeutics or medicalprocedures. In general, each agent will be administered at a dose and/oron a time schedule determined for that agent. In some embodiments, thedisclosure encompasses the delivery of pharmaceutical, prophylactic,diagnostic, or imaging compositions in combination with agents thatimprove their bioavailability, reduce and/or modify their metabolism,inhibit their excretion, and/or modify their distribution within thebody. In certain embodiments, provided are combination therapeuticscontaining one or more modified nucleic acids containing translatableregions that encode for a protein or proteins that boost a mammaliansubject's immunity along with a protein that induces antibody-dependentcellular toxicity. For example, provided are therapeutics containing oneor more nucleic acids that encode trastuzumab and granulocyte-colonystimulating factor (G-CSF). In particular, such combination therapeuticsare useful in Her2+ breast cancer patients who develop inducedresistance to trastuzumab. (See, e.g., Albrecht, Immunotherapy.2(6):795-8 (2010)).

It will further be appreciated that therapeutically, prophylactically,diagnostically, or imaging active agents utilized in combination may beadministered together in a single composition or administered separatelyin different compositions. In general, it is expected that agentsutilized in combination with be utilized at levels that do not exceedthe levels at which they are utilized individually. In some embodiments,the levels utilized in combination will be lower than those utilizedindividually.

The particular combination of therapies (therapeutics or procedures) toemploy in a combination regimen will take into account compatibility ofthe desired therapeutics and/or procedures and the desired therapeuticeffect to be achieved. It will also be appreciated that the therapiesemployed may achieve a desired effect for the same disorder (forexample, a composition useful for treating cancer in accordance with thedisclosure may be administered concurrently with a chemotherapeuticagent), or they may achieve different effects (e.g., control of anyadverse effects).

Kits.

The disclosure provides a variety of kits for conveniently and/oreffectively carrying out methods of the present disclosure. For example,described herein are kits for protein production using a modifiednucleic acid described herein. Typically kits will comprise sufficientamounts and/or numbers of components to allow a user to perform multipletreatments of a subject(s) and/or to perform multiple experiments.

DEFINITIONS

Therapeutic Agent: The term “therapeutic agent” refers to any agentthat, when administered to a subject, has a therapeutic, diagnostic,and/or prophylactic effect and/or elicits a desired biological and/orpharmacological effect.

Animal: As used herein, the term “animal” refers to any member of theanimal kingdom. In some embodiments, “animal” refers to humans at anystage of development. In some embodiments, “animal” refers to non-humananimals at any stage of development. In certain embodiments, thenon-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit,a monkey, a dog, a cat, a sheep, cattle, a primate, or a pig). In someembodiments, animals include, but are not limited to, mammals, birds,reptiles, amphibians, fish, and worms. In some embodiments, the animalis a transgenic animal, genetically-engineered animal, or a clone.

Approximately: As used herein, the term “approximately” or “about,” asapplied to one or more values of interest, refers to a value that issimilar to a stated reference value. In certain embodiments, the term“approximately” or “about” refers to a range of values that fall within25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%,6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than orless than) of the stated reference value unless otherwise stated orotherwise evident from the context (except where such number wouldexceed 100% of a possible value).

Associated with: As used herein, the terms “associated with,”“conjugated,” “linked,” “attached,” and “tethered,” when used withrespect to two or more moieties, means that the moieties are physicallyassociated or connected with one another, either directly or via one ormore additional moieties that serves as a linking agent, to form astructure that is sufficiently stable so that the moieties remainphysically associated under the conditions in which the structure isused, e.g., physiological conditions.

Biologically active: As used herein, the phrase “biologically active”refers to a characteristic of any substance that has activity in abiological system and/or organism. For instance, a substance that, whenadministered to an organism, has a biological effect on that organism,is considered to be biologically active. In particular embodiments,where a nucleic acid is biologically active, a portion of that nucleicacid that shares at least one biological activity of the whole nucleicacid is typically referred to as a “biologically active” portion.

Conserved: As used herein, the term “conserved” refers to nucleotides oramino acid residues of a polynucleotide sequence or amino acid sequence,respectively, that are those that occur-unaltered in the same positionof two or more related sequences being compared. Nucleotides or aminoacids that are relatively conserved are those that are conserved amongstmore related sequences than nucleotides or amino acids appearingelsewhere in the sequences. In some embodiments, two or more sequencesare said to be “completely conserved” if they are 100% identical to oneanother. In some embodiments, two or more sequences are said to be“highly conserved” if they are at least 70% identical, at least 80%identical, at least 90% identical, or at least 95% identical to oneanother. In some embodiments, two or more sequences are said to be“highly conserved” if they are about 70% identical, about 80% identical,about 90% identical, about 95%, about 98%, or about 99% identical to oneanother. In some embodiments, two or more sequences are said to be“conserved” if they are at least 30% identical, at least 40% identical,at least 50% identical, at least 60% identical, at least 70% identical,at least 80% identical, at least 90% identical, or at least 95%identical to one another. In some embodiments, two or more sequences aresaid to be “conserved” if they are about 30% identical, about 40%identical, about 50% identical, about 60% identical, about 70%identical, about 80% identical, about 90% identical, about 95%identical, about 98% identical, or about 99% identical to one another.

Expression: As used herein, “expression” of a nucleic acid sequencerefers to one or more of the following events: (1) production of an RNAtemplate from a DNA sequence (e.g., by transcription); (2) processing ofan RNA transcript (e.g., by splicing, editing, 5′ cap formation, and/or3′ end processing); (3) translation of an RNA into a polypeptide orprotein; and (4) post-translational modification of a polypeptide orprotein.

Ex vivo: As used herein, “ex vivo” refers to events that which occuroutside an organism, e.g., in or on tissue in an artificial environmentoutside the organism, e.g., with the minimum alteration of naturalconditions.

Functional: As used herein, a “functional” biological molecule is abiological molecule in a form in which it exhibits a property and/oractivity by which it is characterized.

Homology: As used herein, the term “homology” refers to the overallrelatedness between polymeric molecules, e.g. between nucleic acidmolecules (e.g. DNA molecules and/or RNA molecules) and/or betweenpolypeptide molecules. In some embodiments, polymeric molecules areconsidered to be “homologous” to one another if their sequences are atleast 25%, at least 30%, at least 35%, at least 40%, at least 45%, atleast 50%, at least 55%, at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, or atleast 99% identical. In some embodiments, polymeric molecules areconsidered to be “homologous” to one another if their sequences are atleast 25%, at least 30%, at least 35%, at least 40%, at least 45%, atleast 50%, at least 55%, at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, or atleast 99% similar. The term “homologous” necessarily refers to acomparison between at least two sequences (nucleotides sequences oramino acid sequences). In accordance with the disclosure, two nucleotidesequences are considered to be homologous if the polypeptides theyencode are at least about 50% identical, at least about 60% identical,at least about 70% identical, at least about 80% identical, or at leastabout 90% identical for at least one stretch of at least about 20 aminoacids. In some embodiments, homologous nucleotide sequences arecharacterized by the ability to encode a stretch of at least 4-5uniquely specified amino acids. Both the identity and the approximatespacing of these amino acids relative to one another must be consideredfor nucleotide sequences to be considered homologous. For nucleotidesequences less than 60 nucleotides in length, homology is determined bythe ability to encode a stretch of at least 4-5 uniquely specified aminoacids. In accordance with the disclosure, two protein sequences areconsidered to be homologous if the proteins are at least about 50%identical, at least about 60% identical, at least about 70% identical,at least about 80% identical, or at least about 90% identical for atleast one stretch of at least about 20 amino acids.

Identity: As used herein, the term “identity” refers to the overallrelatedness between polymeric molecules, e.g., between nucleic acidmolecules (e.g. DNA molecules and/or RNA molecules) and/or betweenpolypeptide molecules. Calculation of the percent identity of twonucleic acid sequences, for example, can be performed by aligning thetwo sequences for optimal comparison purposes (e.g., gaps can beintroduced in one or both of a first and a second nucleic acid sequencesfor optimal alignment and non-identical sequences can be disregarded forcomparison purposes). In certain embodiments, the length of a sequencealigned for comparison purposes is at least 30%, at least 40%, at least50%, at least 60%, at least 70%, at least 80%, at least 90%, at least95%, or 100% of the length of the reference sequence. The nucleotides atcorresponding nucleotide positions are then compared. When a position inthe first sequence is occupied by the same nucleotide as thecorresponding position in the second sequence, then the molecules areidentical at that position. The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences, taking into account the number of gaps, and the length ofeach gap, which needs to be introduced for optimal alignment of the twosequences. The comparison of sequences and determination of percentidentity between two sequences can be accomplished using a mathematicalalgorithm. For example, the percent identity between two nucleotidesequences can be determined using methods such as those described inComputational Molecular Biology, Lesk, A. M., ed., Oxford UniversityPress, New York, 1988; Biocomputing: Informatics and Genome Projects,Smith, D. W., ed., Academic Press, New York, 1993; Sequence Analysis inMolecular Biology, von Heinje, G., Academic Press, 1987; ComputerAnalysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G.,eds., Humana Press, New Jersey, 1994; and Sequence Analysis Primer,Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991;each of which is incorporated herein by reference. For example, thepercent identity between two nucleotide sequences can be determinedusing the algorithm of Meyers and Miller (CABIOS, 1989, 4:11-17), whichhas been incorporated into the ALIGN program (version 2.0) using aPAM120 weight residue table, a gap length penalty of 12 and a gappenalty of 4. The percent identity between two nucleotide sequences can,alliteratively, be determined using the GAP program in the GCG softwarepackage using an NWSgapdna.CMP matrix. Methods commonly employed todetermine percent identity between sequences include, but are notlimited to those disclosed in Carillo, H., and Lipman, D., SIAM JApplied Math., 48:1073 (1988); incorporated herein by reference.Techniques for determining identity are codified in publicly availablecomputer programs. Exemplary computer software to determine homologybetween two sequences include, but are not limited to, GCG programpackage, Devereux, J., et al., Nucleic Acids Research, 12(1), 387(1984)), BLASTP, BLASTN, and FASTA Atschul, S. F. et al., J. MoZee.Bioi., 215, 403 (1990)).

Inhibit expression of a gene: As used herein, the phrase “inhibitexpression of a gene” means to cause a reduction in the amount of anexpression product of the gene. The expression product can be an RNAtranscribed from the gene (e.g., an mRNA) or a polypeptide translatedfrom an mRNA transcribed from the gene. Typically a reduction in thelevel of an mRNA results in a reduction in the level of a polypeptidetranslated therefrom. The level of expression may be determined usingstandard techniques for measuring mRNA or protein.

In vitro: As used herein, the term “in vitro” refers to events thatoccur in an artificial environment, e.g., in a test tube or reactionvessel, in cell culture, in a Petri dish, etc., rather than within anorganism (e.g., animal, plant, or microbe).

In vivo: As used herein, the term “in vivo” refers to events that occurwithin an organism (e.g., animal, plant, or microbe).

Isolated: As used herein, the term “isolated” refers to a substance orentity that has been (1) separated from at least some of the componentswith which it was associated when initially produced (whether in natureor in an experimental setting), and/or (2) produced, prepared, and/ormanufactured by the hand of man. Isolated substances and/or entities maybe separated from at least about 10%, about 20%, about 30%, about 40%,about 50%, about 60%, about 70%, about 80%, about 90%, or more of theother components with which they were initially associated. In someembodiments, isolated agents are more than about 80%, about 85%, about90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%,about 97%, about 98%, about 99%, or more than about 99% pure. As usedherein, a substance is “pure” if it is substantially free of othercomponents.

Preventing: As used herein, the term “preventing” refers to partially orcompletely delaying onset of a particular disease, disorder, and/orcondition; partially or completely delaying onset of one or moresymptoms, features, or clinical manifestations of a particular disease,disorder, and/or condition (e.g., prior to an identifiable disease,disorder, and/or condition); partially or completely delayingprogression from a latent disease, disorder, and/or condition to anactive disease, disorder, and/or condition; and/or decreasing the riskof developing pathology associated with a particular disease, disorder,and/or condition.

Similarity: As used herein, the term “similarity” refers to the overallrelatedness between polymeric molecules, e.g. between nucleic acidmolecules (e.g. DNA molecules and/or RNA molecules) and/or betweenpolypeptide molecules. Calculation of percent similarity of polymericmolecules to one another can be performed in the same manner as acalculation of percent identity, except that calculation of percentsimilarity takes into account conservative substitutions as isunderstood in the rut.

Subject: As used herein, the term “subject” or “patient” refers to anyorgan ism to which a composition in accordance with the disclosure maybe administered, e.g., for experimental, diagnostic, prophylactic,and/or therapeutic purposes. Typical subjects include animals (e.g.,mammals such as mice, rats, rabbits, non-human primates, and humans)and/or plants.

Substantially: As used herein, the term “substantially” refers to thequalitative condition of exhibiting total or near-total extent or degreeof a characteristic or property of interest. One of ordinary skill inthe biological arts will understand that biological and chemicalphenomena rarely, if ever, go to completion and/or proceed tocompleteness or achieve or avoid an absolute result. The term“substantially” is therefore used herein to capture the potential lackof completeness inherent in many biological and chemical phenomena.

Suffering from: An individual who is “suffering from” a disease,disorder, and/or condition has been diagnosed with or displays one ormore symptoms of a disease, disorder, and/or condition.

Susceptible to: An individual who is “susceptible to” a disease,disorder, and/or condition has not been diagnosed with and/or may notexhibit symptoms of the disease, disorder, and/or condition. In someembodiments, an individual who is susceptible to a disease, disorder,and/or condition (for example, cancer) may be characterized by one ormore of the following: (1) a genetic mutation associated withdevelopment of the disease, disorder, and/or condition; (2) a geneticpolymorphism associated with development of the disease, disorder,and/or condition; (3) increased and/or decreased expression and/oractivity of a protein and/or nucleic acid associated with the disease,disorder, and/or condition; (4) habits and/or lifestyles associated withdevelopment of the disease, disorder, and/or condition; (5) a familyhistory of the disease, disorder, and/or condition; and (6) exposure toand/or infection with a microbe associated with development of thedisease, disorder, and/or condition. In some embodiments, an individualwho is susceptible to a disease, disorder, and/or condition will developthe disease, disorder, and/or condition. In some embodiments, anindividual who is susceptible to a disease, disorder, and/or conditionwill not develop the disease, disorder, and/or condition.

Therapeutically effective amount: As used herein, the term“therapeutically effective amount” means an amount of an agent to bedelivered (e.g., nucleic acid, drug, therapeutic agent, diagnosticagent, prophylactic agent, etc.) that is sufficient, when administeredto a subject suffering from or susceptible to a disease, disorder,and/or condition, to treat, improve symptoms of, diagnose, prevent,and/or delay the onset of the disease, disorder, and/or condition.

Transcription factor: As used herein, the term “transcription factor”refers to a DNA-binding protein that regulates transcription of DNA intoRNA, for example, by activation or repression of transcription. Sometranscription factors effect regulation of transcription alone, whileothers act in concert with other proteins. Some transcription factor canboth activate and repress transcription under certain conditions. Ingeneral, transcription factors bind a specific target sequence orsequences highly similar to a specific consensus sequence in aregulatory region of a target gene. Transcription factors may regulatetranscription of a target gene alone or in a complex with othermolecules.

Treating: As used herein, the term “treating” refers to partially orcompletely alleviating, ameliorating, improving, relieving, delayingonset of, inhibiting progression of, reducing severity of, and/orreducing incidence of one or more symptoms, features, or clinicalmanifestations of a particular disease, disorder, and/or condition. Forexample, “treating” cancer may refer to inhibiting survival, growth, nild/or spread of a tumor. Treatment may be administered to a subject whodoes not exhibit signs of a disease, disorder, and/or condition (e.g.,prior to an identifiable disease, disorder, and/or condition), and/or toa subject who exhibits only early signs of a disease, disorder, and/orcondition for the purpose of decreasing the risk of developing pathologyassociated with the disease, disorder, and/or condition. In someembodiments, treatment comprises delivery of a protein associated with atherapeutically active nucleic acid to a subject in need thereof.

Unmodified: As used herein, “unmodified” refers to a nucleic acid priorto being modified.

EXAMPLES Example 1 Synthesis of Modified mRNA

Modified mRNAs (modRNAs) according to the invention were made usingstandard laboratory methods and materials. The open reading frame (ORF)of the gene of interest is flanked by a 5′ untranslated region (UTR)containing a strong Kozak translational initiation signal and analpha-globin 3′ UTR terminating with an oligo(dT) sequence for templatedaddition of a polyA tail. The modRNAs were modified with pseudouridine(ψ) and 5-methyl-cytidine (5meC) to reduce the cellular innate immuneresponse. Kariko K et al. Immunity 23:165-75 (2005), Kariko K et al. MolTher 16:1833-40 (2008), Anderson B R et al. NAR (2010).

The cloning, gene synthesis and vector sequencing was performed byDNA2.0 Inc. (Menlo Park, Calif.). Vector sequences and insert sequencesare set forth in SEQ ID NOs: 5-8. The ORFs were restriction digestedusing XbaI or HindIII and used for cDNA synthesis using tailed-PCR. Thistailed-PCR cDNA product was used as the template for the modified mRNAsynthesis reaction using 25 mM each modified nucleotide mix (modifiedU/C was manufactured by TriLink Biotech, San Diego, Calif., unmodifiedA/G was purchased from Epicenter Biotechnologies, Madison, Wis.) andCellScript MegaScript™ (Epicenter Biotechnologies, Madison, Wis.)complete mRNA synthesis kit. The in vitro transcription reaction was runfor 3-4 hours at 37° C. PCR reaction used HiFi PCR 2× Master Mix™ (KapaBiosystems, Woburn, Mass.). The In vitro transcribed mRNA product wasrun on an agarose gel and visualized. mRNA was purified withAmbion/Applied Biosystems (Austin, Tex.) MEGAClear RNA™ purificationkit. PCR used PureLink™ PCR purification kit (Invitrogen, Carlsbad,Calif.) or PCR cleanup kit (Qiagen, Valencia, Calif.). The product wasquantified on Nanodrop™ UV Absorbance (ThermoFisher, Waltham, Mass.).Quality, UV absorbance quality and visualization of the product wasperformed on an 1.2% agarose gel. The product was resuspended in TEbuffer.

Modified RNAs incorporating adenosine analogs were poly (A) tailed usingyeast Poly (A) Polymerase (Affymettix, Santa Clara, Calif.). PCRreaction used HiFi PCR 2× Master Mix™ (Kapa Biosystems, Woburn, Mass.).Modified RNAs were post-transcriptionally capped using recombinantVaccinia Virus Capping Enzyme (New England BioLabs, Ipswich, Mass.) anda recombinant 2′-o-methyltransferase (Epicenter Biotechnologies,Madison, Wis.) to generate the 5′-guanosine Cap1 structure. Cap 2structure and Cap 3 structure may be generated using additional2′-o-methyltransferases. The in vitro transcribed mRNA product was runon an agarose gel and visualized. Modified RNA was purified withAmbion/Applied Biosystems (Austin, Tex.) MEGAClear RNA™ purificationkit. PCR used PureLink™ PCR purification kit (Invitrogen, Carlsbad,Calif.). The product was quantified on Nanodrop™ UV Absorbance(ThermoFisher, Waltham, Mass.). Quality, UV absorbance quality andvisualization of the product was performed on an 1.2% agarose gel. Theproduct was resuspended in TE buffer.

Exemplary Capping Structures.

5′-capping of modified RNA may be completed concomitantly during the invitro-transcription reaction using the following chemical RNA capanalogs to generate the 5′-guanosine cap structure according tomanufacturer protocols: 3′-O-Me-m7G(5′)ppp(5′)G; G(5′)ppp(5′)A;G(5′)ppp(5′)G; m7G(5′)ppp(5′)A; m7G(5′)ppp(5′)G (New England BioLabs,Ipswich, Mass.). 5′-capping of modified RNA may be completedpost-transcriptionally using a Vaccinia Vims Capping Enzyme to generatethe “Cap 0” structure: m7G(5′)ppp(5′)G (New England BioLabs, Ipswich,Mass.). Cap 1 structure may be generated using both Vaccinia ViJ.usCapping Enzyme and a 2′-0 methyl-transferase to generate:m7G(5′)ppp(5′)G-2′-0-methyl. Cap 2 structure may be generated from theCap 1 structure followed by the 2′-0-methylation of the5′-antepenultimate nucleotide using a 2′-0 methyl-transferase. Cap 3structure may be generated from the Cap 2 structure followed by the2′-O-methylation of the 5′-preantepenultimate nucleotide using a 2′-0methyl-transferase. Enzymes are preferably derived from a recombinantsource.

When transfected into mammalian cells, the modified mRNAs may have astability of between 12-18 hours or more than 18 hours, e.g., 24, 36,48, 60, 72 or greater than 72 hours.

Example 2 De Novo Generation of a Mammalian Commercial Production CellLine Expressing Human G-CSF as a Therapeutic Agent in Model Bioreactor

The nucleic acid sequence for the precursor of human granulocyte colonystimulating factor (G-CSF) is set forth in SEQ ID NO: 1:

(SEQ ID NO: 1) agcttttggaccctcgtacagaagctaatacgactcactatagggaaataagagagaaaagaagagtaagaagaaatataagagccaccatggccggtcccgcgacccaaagccccatgaaacttatggccctgcagttgctgctttggcactcggccctctggacagtccaagaagcgactcctctcggacctgcctcatcgttgccgcagtcattccttttgaagtgtctggagcaggtgcgaaagattcagggcgatggagccgcactccaagagaagctctgcgcgacatacaaactttgccatcccgaggagctcgtactgctcgggcacagcttggggattccctgggctcctctctcgtcctgtccgtcgcaggctttgcagttggcagggtgcctttcccagctccactccggtttgttcttgtatcagggactgctgcaagcccttgagggaatctcgccagaattgggcccgacgctggacacgttgcagctcgacgtggcggatttcgcaacaaccatctggcagcagatggaggaactggggatggcacccgcgctgcagcccacgcagggggcaatgccggcctttgcgtccgcgtttcagcgcagggcgggtggagtcctcgtagcgagccaccttcaatcatttttggaagtctcgtaccgggtgctgagacatcttgcgcagccgtgaagcgctgccttctgcggggcttgccttctggccatgcccttcttctctcccttgcacctgtacctcttggtctttgaataaagcctgagtaggaaggcggccgctcgagcatgcatctagagggcccaattcgccctattcgaagtcg

The nucleic acid sequence for G-CSF mRNA is set forth in SEQ ID NO: 2:

(SEQ ID NO: 2) agcuuuuggacccucguacagaagcuaauacgacucacuauagggaaauaagagagaaaagaagaguaagaagaaauauaagagccaccauggccggucccgcgacccaaagccccaugaaacuuauggcccugcaguugcugcuuuggcacucggcccucuggacaguccaagaagcgacuccucucggaccugccucaucguugccgcagucauuccuuuugaagugucuggagcaggugcgaaagauucagggcgauggagccgcacuccaagagaagcucugcgcgacauacaaacuuugccaucccgaggagcucguacugcucgggcacagcuuggggauucccugggcuccucucucguccuguccgucgcaggcuuugcaguuggcagggugccuuucccagcuccacuccgguuuguucuuguaucagggacugcugcaagcccuugagggaaucucgccagaauugggcccgacgcuggacacguugcagcucgacguggcggauuucgcaacaaccaucuggcagcagauggaggaacuggggauggcacccgcgcugcagcccacgcagggggcaaugccggccuuugcguccgcguuucagcgcagggcggguggaguccucguagcgagccaccuucaaucauuuuuggaagucucguaccgggugcugagacaucuugcgcagccgugaagcgcugccuucugcggggcuugccuucuggccaugcccuucuucucucccuugcaccuguaccucuuggucuuugaauaaagccugaguaggaaggcggccgcucgagcaugcaucuagagggcccaauucgcccuauucgaagucg

The nucleic acid sequence for an exemplary G-CSF modified rnRNA (modRNA)is set forth in SEQ ID NO: 3:

(SEQ ID NO: 3) ag5meCψψψψgga5meC5meC5meCψ5meCgψa5meCagaag5meCψaaψa5meCga5meCψ5meCa5meCψaψagggaaaψaagagagaaaagaagagψaagaagaaaψaψaagag5meC5meCa5meC5meCatψgg5meC5meCggψ5meC5meC5meCg5meCga5meC5meC5meCaaag5meC5meC5meC5meCaψgaaa5meCψψaψgg5meC5meC5meCψg5meCagψψg5meCψg5meCψψψgg5meCa5meCψ5meCgg5meC5meC5meCψ5meCψgga5meCagψ5meC5meCaagaag5meCga5meCψ5meC5meCψ5meCψ5meCgga5meC5meCψg5meC5meCψ5meCaψ5meCgψψg5meC5meCg5meCagψ5meCaψψ5meC5meCψψψψgaagψgψ5meCψggag5meCaggψg5meCgaaagaψψ5meCaggg5meCgaψggag5meC5meCg5meCa5meCψ5meC5meCaagagaag5meCψ5meCψg5meCg5meCga5meCaψa5meCaaa5meCψψψg5meC5meCaψ5meC5meC5meCgaggag5meCψ5meCgψa5meCψg5meCy5meCggg5meCa5meCag5meCψψggggaψψ5meC5meC5meCψggg5meCψ5meC5meCψ5meCψ5meCψ5meCgψ5meC5meCψgψ5meC5meCgψ5meCg5meCagg5meCψψψg5meCagψψgg5meCagggψg5meC5meCψψψ5meC5meC5meCag5meCψ5meC5meCa5meCψ5meC5meCggψψψgψψ5meCψψgψaψ5meCaggga5meCψg5meCψg5meCaag5meC5meC5meCψψgagggaaψ5meCψ5meCg5meC5meCagaaψψggg5meC5meC5meCga5meCg5meCψgga5meCa5meCgψψg5meCag5meCψ5meCga5meCgψgg5meCggaψψψ5meCg5meCaa5meCaa5meC5meCaψ5meCψgg5meCag5meCagaψggaggaa5meCψggggaψgg5meCa5meC5meC5meCg5meCg5meCψg5meCag5meC5meC5meCa5meCg5meCaggggg5meCaaψg5meC5meCgg5meC5meCψψψg5meCgψ5meC5meCg5meCgψψψ5meCag5meCg5meCaggg5meCgggψggagψ5meC5meCψ5meCgψag5meCgag5meC5meCa5meC5meCψψ5meCaatψ5meCaψψψψψggaagψ5meCψ5meCgψa5meC5meCgggψg5meCψgaga5meCaψ5meCψψg5meCg5meCag5meC5meCgψgaag5meCg5meCψg5meC5meCψψ5meCψg5meCgggg5meCψψg5meC5meCψψ5meCψgg5meC5meCaψg5meC5meC5meCψψ5meCψψ5meCψ5meCψ5meC5meC5meCψψg5meCa5meC5meCψgψa5meC5meCψ5meCψψggψ5meCψψψgaaψaaag5meC5meCψgagψaggaagg5meCgg5meC5meCg5meCψ5meCgag5meCaψg5meCaψ5meCψagaggg5meC5meC5meCaaψψ5meCg5meC5meC5meCψaψψψ5me Cgaagψ5meCg

FIG. 1 shows an Enzyme-linked immunosorbent assay (ELISA) for HumanGranulocyte-Colony Stimulating Factor (G-CSF) from Chinese Hamster OvaryCells (CHO) transfected with modRNA for G-CSF. The CHO cells were grownin CD CHO Medium with Supplement of L-Glutamine, Hypoxanthine andThymidine. 2×10₆ Cells were transfected with 24 ug modRNA complexed withRNAiMax from Invitrogen in a 75 cm₂ culture flask from Corning with 7 mlof medium. The RNA:RNAiMAX complex was formed by first incubating theRNA with CD CHO Medium in a 5× volumetric dilution for 10 minutes atroom temperature. In a second vial, RNAiMAX reagent was incubated withCD CHO Medium in a 10× volumetric dilution for 10 minutes at roomtemperature. The RNA vial was then mixed with the RNAiMAX vial andincubated for 20-30 at room temperature before being added to the cellsin a drop-wise fashion. The concentration of secreted huG-CSF in theculture medium was measured at 12 and 24 hours post-transfection. Cellsupernatants were stored at −20° C. Secretion of HumanGranulocyte-Colony Stimulating Factor (G-CSF) from transfected HumanEmbryonic Kidney cells was quantified using an ELISA kit from Invitrogenfollowing the manufacturers recommended instructions. These data showthat huG-CSF modRNA (SEQ ID NO: 3) is capable of being translated in CHOcells, and that huG-CSF is secreted out of the cells and released intothe extracellular environment. Furthermore these data demonstrate thattransfection of cells with modRNA huG-CSF for the production of secretedprotein can be scaled up to a bioreactor or large cell cultureconditions.

Example 3 De Novo Generation of a Mammalian Commercial Production CellLine Expressing Humanized IgG Antibodies (Trastuzumab and Rituximab) asa Therapeutic Agent in Model Bioreactor

The nucleic acid sequence for the Heavy Chain of Rituximab is set forthin SEQ ID NO: 4:

(SEQ ID NO: 4) CTCGTACAGAAGCTAATACGACTCACTATAGGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAG AGCCACCATGGCCGTGATGGCGCCGAGGACCCTGGTGCTCTTGCTCACGGGTGCCTTGGCCCTCACGCAA ACATGGGCGGGACAGGCGTACTTGCAGCAGTCAGGGGCAGAACTCGTAAGGCCCGGAGCGTCGGTGAAGA TGTCGTGTAAAGCGTCGGGCTATACTTTCACATCGTACAACATGCACTGGGTCAAACAGACGCCCCGACA AGGGCTGGAGTGGATTGGAGCTATCTACCCCGGTAACGGGGATACGTCGTACAACCAGAAGTTTAAGGGG AAGGCGACTCTTACTGTCGACAAGTCGTCCTCCACCGCCTATATGCAGCTGTCGAGCCTGACTTCGGAAG ATTCAGCGGTGTACTTTTGTGCGCGCGTGGTCTATTACTCAAATTCGTATTGGTATTTCGATGTGTGGGG TACGGGGACCACTGTGACCGTGTCAGGACCCTCGGTATTCCCCCTCGCGCCTAGCTCAAAGTCCACCTCC GGGGGAACAGCCGCCTTGGGTTGCTTGGTAAAGGACTATTTCCCCGAGCCCGTCACAGTGAGCTGGAACT CCGGGGCACTGACATCGGGAGTGCACACGTTTCCCGCGGTACTTCAGTCATCAGGACTCTACTCGCTGTC AAGCGTGGTCACGGTGCCTTCATCCTCCCTTGGAACGCAGACTTACATCTGCAACGTGAATCATAAGCCT AGCAATACCAAGGTCGACAAGAAAGCCGAACCCAAATCATGTGATAAAACACACACGTGTCCTCCCTGCC CCGCACCGGAGCTTCTCGGGGGACCGAGCGTGTTCTTGTTTCCACCTAAGCCGAAAGATACGCTTATGAT CTCCCGGACCCCCGAAGTAACTTGCGTAGTAGTAGACGTAAGCCACGAGGACCCCGAAGTGAAATTCAAT TGGTACGTCGACGGAGTGGAGGTCCATAATGCGAAAACAAAGCCGAGAGAGGAACAGTACAATTCCACAT ACCGCGTCGTAAGCGTCTTGACAGTATTGCATCAGGATTGGCTGAACGGAAAGGAATACAAGTGCAAAGT ATCAAACAAAGCACTTCCGGCACCGATTGAAAAGACGATCTCAAAAGCAAAAGGGCAACCTCGGGAGCCA CAAGTCTATACTCTCCCGCCGTCGCGCGATGAATTGACCAAAAACCAGGTGTCCCTTACATGTCTCGTAA AGGGTTTTTACCCGTCAGACATCGCCGTCGAGTGGGAGTCAAACGGTCAGCCGGAGAATAACTATAAGAC GACCCCACCAGTCTTGGACAGCGATGGCTCCTTCTTCTTGTATTCAAAGCTGACGGTGGACAAATCGAGA TGGCAGCAGGGTAATGTGTTTTCGTGCAGCGTCATGCACGAGGCGCTTCATAATCATTACACTCAAAAGT CCCTGTCGCTGTCGCCCGGAAAGCACCATCACCACCACCATTGAAGCGCTGCCTTCTGCGGGGCTTGCCT TCTGGCCATGCCCTTCTTCTCTCCCTTGCACCTGTACCTCTTGGTCTTTGAATAAAGCCTGAGTAGGAAG GCGGCCGCTCGAGCATGCATCTAGA

The nucleic acid sequence for the mRNA for the Heavy Chain of Rituximabis set forth in SEQ ID NO: 5:

(SEQ ID NO: 5) CUCGUACAGAAGCUAAUACGACUCACUAUAGGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAG AGCCACCAUGGCCGUGAUGGCGCCGAGGACCCUGGUGCUCUUGCUCACGGGUGCCUUGGCCCUCACGCAA ACAUGGGCGGGACAGGCGUACUUGCAGCAGUCAGGGGCAGAACUCGUAAGGCCCGGAGCGUCGGUGAAGA UGUCGUGUAAAGCGUCGGGCUAUACUUUCACAUCGUACAACAUGCACUGGGUCAAACAGACGCCCCGACA AGGGCUGGAGUGGAUUGGAGCUAUCUACCCCGGUAACGGGGAUACGUCGUACAACCAGAAGUUUAAGGGG AAGGCGACUCUUACUGUCGACAAGUCGUCCUCCACCGCCUAUAUGCAGCUGUCGAGCCUGACUUCGGAAG AUUCAGCGGUGUACUUUUGUGCGCGCGUGGUCUAUUACUCAAAUUCGUAUUGGUAUUUCGAUGUGUGGGG UACGGGGACCACUGUGACCGUGUCAGGACCCUCGGUAUUCCCCCUCGCGCCUAGCUCAAAGUCCACCUCC GGGGGAACAGCCGCCUUGGGUUGCUUGGUAAAGGACUAUUUCCCCGAGCCCGUCACAGUGAGCUGGAACU CCGGGGCACUGACAUCGGGAGUGCACACGUUUCCCGCGGUACUUCAGUCAUCAGGACUCUACUCGCUGUC AAGCGUGGUCACGGUGCCUUCAUCCUCCCUUGGAACGCAGACUUACAUCUGCAACGUGAAUCAUAAGCCU AGCAAUACCAAGGUCGACAAGAAAGCCGAACCCAAAUCAUGUGAUAAAACACACACGUGUCCUCCCUGCC CCGCACCGGAGCUUCUCGGGGGACCGAGCGUGUUCUUGUUUCCACCUAAGCCGAAAGAUACGCUUAUGAU CUCCCGGACCCCCGAAGUAACUUGCGUAGUAGUAGACGUAAGCCACGAGGACCCCGAAGUGAAAUUCAAU UGGUACGUCGACGGAGUGGAGGUCCAUAAUGCGAAAACAAAGCCGAGAGAGGAACAGUACAAUUCCACAU ACCGCGUCGUAAGCGUCUUGACAGUAUUGCAUCAGGAUUGGCUGAACGGAAAGGAAUACAAGUGCAAAGU AUCAAACAAAGCACUUCCGGCACCGAUUGAAAAGACGAUCUCAAAAGCAAAAGGGCAACCUCGGGAGCCA CAAGUCUAUACUCUCCCGCCGUCGCGCGAUGAAUUGACCAAAAACCAGGUGUCCCUUACAUGUCUCGUAA AGGGUUUUUACCCGUCAGACAUCGCCGUCGAGUGGGAGUCAAACGGUCAGCCGGAGAAUAACUAUAAGAC GACCCCACCAGUCUUGGACAGCGAUGGCUCCUUCUUCUUGUAUUCAAAGCUGACGGUGGACAAAUCGAGA UGGCAGCAGGGUAAUGUGUUUUCGUGCAGCGUCAUGCACGAGGCGCUUCAUAAUCAUUACACUCAAAAGU CCCUGUCGCUGUCGCCCGGAAAGCACCAUCACCACCACCAUUGAAGCGCUGCCUUCUGCGGGGCUUGCCU UCUGGCCAUGCCCUUCUUCUCUCCCUUGCACCUGUACCUCUUGGUCUUUGAAUAAAGCCUGAGUAGGAAG GCGGCCGCUCGAGCAUGCAUCUAGA

The nucleic acid sequence for the nucleic acid sequence for the LightChain of Rituximab is set forth in SEQ ID NO: 6:

(SEQ ID NO: 6) CTCGTACAGAAGCTAATACGACTCACTATAGGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAG AGCCACCATGGCTGTCATGGCCCCGAGAACACTTGTGCTGTTGTTGACAGGAGCGCTCGCACTCACACAG ACTTGGGCCGGTCAGATTGTGCTCAGCCAGTCGCCAGCGATCCTTTCGGCCTCCCCTGGTGAGAAAGTAA CGATGACGTGCCGAGCCTCCTCAAGCGTGTCATACATGCATTGGTATCAGCAGAAGCCTGGGTCGTCGCC CAAGCCCTGGATCTACGCCCCGTCCAATCTTGCGTCAGGGGTCCCGGCACGGTTCAGCGGATCGGGGTCG GGTACATCGTATTCACTCACGATTAGCCGCGTAGAGGCCGAGGACGCGGCGACTTACTACTGTCAGCAAT GGTCCTTTAATCCACCCACGTTTGGAGCGGGCACCAAGCTCGAACTTAAAAGAACGGTCGCCGCACCCTC AGTGTTTATCTTCCCGCCCTCGGACGAACAACTTAAGTCGGGGACCGCTTCCGTGGTGTGCTTGCTGAAC AATTTCTATCCTCGGGAAGCTAAAGTGCAATGGAAAGTCGATAACGCATTGCAGAGCGGAAACTCACAAG AGTCGGTAACTGAGCAGGATAGCAAGGATTCGACATACTCGCTGAGCAGCACGCTGACGTTGTCCAAGGC GGACTACGAGAAACACAAGGTATATGCGTGTGAAGTCACCCACCAGGGATTGTCATCGCCGGTCACCAAA TCATTCAACAGGTGATAAAGCGCTGCCTTCTGCGGGGCTTGCCTTCTGGCCATGCCCTTCTTCTCTCCCT TGCACCTGTACCTCTTGGTCTTTGAATAAAGCCTGAGTAGGAAGGCGGCCGCTCGAGCATGCATCTAGA

The nucleic acid sequence for the mRNA of the Light Chain of Rituximabis set forth in SEQ ID NO: 7:

(SEQ ID NO: 7) CUCGUACAGAAGCUAAUACGACUCACUAUAGGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAG AGCCACCAUGGCUGUCAUGGCCCCGAGAACACUUGUGCUGUUGUUGACAGGAGCGCUCGCACUCACACAG ACUUGGGCCGGUCAGAUUGUGCUCAGCCAGUCGCCAGCGAUCCUUUCGGCCUCCCCUGGUGAGAAAGUAA CGAUGACGUGCCGAGCCUCCUCAAGCGUGUCAUACAUGCAUUGGUAUCAGCAGAAGCCUGGGUCGUCGCC CAAGCCCUGGAUCUACGCCCCGUCCAAUCUUGCGUCAGGGGUCCCGGCACGGUUCAGCGGAUCGGGGUCG GGUACAUCGUAUUCACUCACGAUUAGCCGCGUAGAGGCCGAGGACGCGGCGACUUACUACUGUCAGCAAU GGUCCUUUAAUCCACCCACGUUUGGAGCGGGCACCAAGCUCGAACUUAAAAGAACGGUCGCCGCACCCUC AGUGUUUAUCUUCCCGCCCUCGGACGAACAACUUAAGUCGGGGACCGCUUCCGUGGUGUGCUUGCUGAAC AAUUUCUAUCCUCGGGAAGCUAAAGUGCAAUGGAAAGUCGAUAACGCAUUGCAGAGCGGAAACUCACAAG AGUCGGUAACUGAGCAGGAUAGCAAGGAUUCGACAUACUCGCUGAGCAGCACGCUGACGUUGUCCAAGGC GGACUACGAGAAACACAAGGUAUAUGCGUGUGAAGUCACCCACCAGGGAUUGUCAUCGCCGGUCACCAAA UCAUUCAACAGGUGAUAAAGCGCUGCCUUCUGCGGGGCUUGCCUUCUGGCCAUGCCCUUCUUCUCUCCCU UGCACCUGUACCUCUUGGUCUUUGAAUAAAGCCUGAGUAGGAAGGCGGCCGCUCGAGCAUGCAUCUAGA

The nucleic acid sequence for the nucleic acid sequence for the HeavyChain of Trastuzumab is set forth in SEQ ID NO: 8:

(SEQ ID NO: 8) CTCGTACAGAAGCTAATACGACTCACTATAGGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAG AGCCACCATGGCCGTGATGGCGCCGCGGACCCTGGTCCTCCTGCTGACCGGCGCCCTCGCCCTGACGCAG ACCTGGGCCGGGGAGGTGCAGCTGGTCGAGAGCGGCGGGGGCCTCGTGCAGCCGGGCGGGTCGCTGCGGC TGAGCTGCGCCGCGAGCGGGTTCAACATCAAGGACACCTACATCCACTGGGTGCGCCAGGCCCCCGGCAA GGGCCTCGAGTGGGTCGCCCGGATCTACCCCACGAACGGGTACACCCGCTACGCCGACAGCGTGAAGGGC CGGTTCACCATCAGCGCGGACACCTCGAAGAACACGGCCTACCTGCAGATGAACAGCCTGCGCGCCGAGG ACACCGCCGTGTACTACTGCAGCCGGTGGGGCGGCGACGGGTTCTACGCCATGGACTACTGGGGGCAGGG CACCCTCGTCACCGTGAGCAGCGCGTCGACGAAGGGGCCCAGCGTGTTCCCGCTGGCCCCCAGCAGCAAG AGCACCAGCGGCGGGACCGCCGCCCTGGGCTGCCTCGTCAAGGACTACTTCCCCGAGCCCGTGACCGTGT CGTGGAACAGCGGCGCGCTGACGAGCGGGGTCCACACCTTCCCGGCCGTGCTGCAGAGCAGCGGCCTCTA CTCGCTGAGCAGCGTGGTCACCGTGCCCAGCAGCAGCCTGGGGACCCAGACGTACATCTGCAACGTGAAC CACAAGCCCTCGAACACCAAGGTCGACAAGAAGGTGGAGCCCCCGAAGAGCTGCGACAAGACCCACACCT GCCCGCCCTGCCCCGCCCCCGAGCTCCTGGGCGGGCCCAGCGTGTTCCTGTTCCCGCCCAAGCCCAAGGA CACGCTCATGATCAGCCGCACCCCCGAGGTCACCTGCGTGGTGGTCGACGTGAGCCACGAGGACCCCGAG GTGAAGTTCAACTGGTACGTCGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCGCGGGAGGAGCAGT ACAACTCGACGTACCGCGTCGTGAGCGTGCTGACCGTCCTGCACCAGGACTGGCTCAACGGCAAGGAGTA CAAGTGCAAGGTGAGCAACAAGGCCCTGCCCGCGCCCATCGAGAAGACCATCAGCAAGGCCAAGGGGCAG CCCCGGGAGCCGCAGGTGTACACCCTGCCCCCCAGCCGCGACGAGCTCACGAAGAACCAGGTCAGCCTGA CCTGCCTGGTGAAGGGCTTCTACCCCTCGGACATCGCCGTGGAGTGGGAGAGCAACGGGCAGCCGGAGAA CAACTACAAGACCACCCCGCCCGTCCTCGACAGCGACGGCAGCTTCTTCCTGTACAGCAAGCTGACGGTG GACAAGTCGCGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTCATGCACGAGGCCCTCCACAACCACT ACACCCAGAAGAGCCTGAGCCTGAGCCCCGGGAAGCATCATCATCATCATCATTGAAGCGCTGCCTTCTG CGGGGCTTGCCTTCTGGCCATGCCCTTCTTCTCTCCCTTGCACCTGTACCTCTTGGTCTTTGAATAAAGC CTGAGTAGGAAGGCGGCCGCTCGAGCATGCATCTAGA

The nucleic acid sequence of the mRNA for the Heavy Chain of Trastuzumabis set forth in SEQ ID NO: 9:

(SEQ ID NO: 9) CUCGUACAGAAGCUAAUACGACUCACUAUAGGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAG AGCCACCAUGGCCGUGAUGGCGCCGCGGACCCUGGUCCUCCUGCUGACCGGCGCCCUCGCCCUGACGCAG ACCUGGGCCGGGGAGGUGCAGCUGGUCGAGAGCGGCGGGGGCCUCGUGCAGCCGGGCGGGUCGCUGCGGC UGAGCUGCGCCGCGAGCGGGUUCAACAUCAAGGACACCUACAUCCACUGGGUGCGCCAGGCCCCCGGCAA GGGCCUCGAGUGGGUCGCCCGGAUCUACCCCACGAACGGGUACACCCGCUACGCCGACAGCGUGAAGGGC CGGUUCACCAUCAGCGCGGACACCUCGAAGAACACGGCCUACCUGCAGAUGAACAGCCUGCGCGCCGAGG ACACCGCCGUGUACUACUGCAGCCGGUGGGGCGGCGACGGGUUCUACGCCAUGGACUACUGGGGGCAGGG CACCCUCGUCACCGUGAGCAGCGCGUCGACGAAGGGGCCCAGCGUGUUCCCGCUGGCCCCCAGCAGCAAG AGCACCAGCGGCGGGACCGCCGCCCUGGGCUGCCUCGUCAAGGACUACUUCCCCGAGCCCGUGACCGUGU CGUGGAACAGCGGCGCGCUGACGAGCGGGGUCCACACCUUCCCGGCCGUGCUGCAGAGCAGCGGCCUCUA CUCGCUGAGCAGCGUGGUCACCGUGCCCAGCAGCAGCCUGGGGACCCAGACGUACAUCUGCAACGUGAAC CACAAGCCCUCGAACACCAAGGUCGACAAGAAGGUGGAGCCCCCGAAGAGCUGCGACAAGACCCACACCU GCCCGCCCUGCCCCGCCCCCGAGCUCCUGGGCGGGCCCAGCGUGUUCCUGUUCCCGCCCAAGCCCAAGGA CACGCUCAUGAUCAGCCGCACCCCCGAGGUCACCUGCGUGGUGGUCGACGUGAGCCACGAGGACCCCGAG GUGAAGUUCAACUGGUACGUCGACGGCGUGGAGGUGCACAACGCCAAGACCAAGCCGCGGGAGGAGCAGU ACAACUCGACGUACCGCGUCGUGAGCGUGCUGACCGUCCUGCACCAGGACUGGCUCAACGGCAAGGAGUA CAAGUGCAAGGUGAGCAACAAGGCCCUGCCCGCGCCCAUCGAGAAGACCAUCAGCAAGGCCAAGGGGCAG CCCCGGGAGCCGCAGGUGUACACCCUGCCCCCCAGCCGCGACGAGCUCACGAAGAACCAGGUCAGCCUGA CCUGCCUGGUGAAGGGCUUCUACCCCUCGGACAUCGCCGUGGAGUGGGAGAGCAACGGGCAGCCGGAGAA CAACUACAAGACCACCCCGCCCGUCCUCGACAGCGACGGCAGCUUCUUCCUGUACAGCAAGCUGACGGUG GACAAGUCGCGGUGGCAGCAGGGCAACGUGUUCAGCUGCAGCGUCAUGCACGAGGCCCUCCACAACCACU ACACCCAGAAGAGCCUGAGCCUGAGCCCCGGGAAGCAUCAUCAUCAUCAUCAUUGAAGCGCUGCCUUCUG CGGGGCUUGCCUUCUGGCCAUGCCCUUCUUCUCUCCCUUGCACCUGUACCUCUUGGUCUUUGAAUAAAGC CUGAGUAGGAAGGCGGCCGCUCGAGCAUGCAUCUAGA

The nucleic acid sequence for the nucleic acid sequence for the LightChain of Trastuzumab is set forth in SEQ ID NO: 10:

(SEQ ID NO: 10) CTCGTACAGAAGCTAATACGACTCACTATAGGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAG AGCCACCATGGCCGTGATGGCGCCGCGGACCCTGGTCCTCCTGCTGACCGGCGCCCTCGCCCTGACGCAG ACCTGGGCCGGGGACATCCAGATGACCCAGAGCCCGTCGAGCCTGAGCGCCAGCGTGGGCGACCGGGTCA CGATCACCTGCCGCGCGAGCCAGGACGTGAACACCGCCGTGGCCTGGTACCAGCAGAAGCCCGGGAAGGC CCCCAAGCTCCTGATCTACTCGGCGAGCTTCCTGTACAGCGGCGTCCCCAGCCGGTTCAGCGGGTCGCGC AGCGGCACCGACTTCACGCTCACCATCAGCAGCCTGCAGCCGGAGGACTTCGCCACCTACTACTGCCAGC AGCACTACACCACGCCCCCCACCTTCGGGCAGGGCACCAAGGTGGAGATCAAGCGGACCGTGGCCGCCCC CAGCGTCTTCATCTTCCCGCCCAGCGACGAGCAGCTGAAGTCGGGCACGGCCAGCGTGGTGTGCCTCCTG AACAACTTCTACCCCCGCGAGGCGAAGGTCCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGGAACAGCC AGGAGAGCGTGACCGAGCAGGACTCGAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAA GGCCGACTACGAGAAGCACAAGGTCTACGCCTGCGAGGTGACCCACCAGGGGCTCTCGAGCCCCGTGACC AAGAGCTTCAACCGGGGCGAGTGCTGAAGCGCTGCCTTCTGCGGGGCTTGCCTTCTGGCCATGCCCTTCT TCTCTCCCTTGCACCTGTACCTCTTGGTCTTTGAATAAAGCCTGAGTAGGAAGGCGGCCGCTCGAGCATG CATCTAGA

The nucleic acid sequence for the mRNA of the Light Chain of Trastuzumabis set forth in SEQ ID NO: 11:

(SEQ ID NO: 11) CUCGUACAGAAGCUAAUACGACUCACUAUAGGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAG AGCCACCAUGGCCGUGAUGGCGCCGCGGACCCUGGUCCUCCUGCUGACCGGCGCCCUCGCCCUGACGCAG ACCUGGGCCGGGGACAUCCAGAUGACCCAGAGCCCGUCGAGCCUGAGCGCCAGCGUGGGCGACCGGGUCA CGAUCACCUGCCGCGCGAGCCAGGACGUGAACACCGCCGUGGCCUGGUACCAGCAGAAGCCCGGGAAGGC CCCCAAGCUCCUGAUCUACUCGGCGAGCUUCCUGUACAGCGGCGUCCCCAGCCGGUUCAGCGGGUCGCGC AGCGGCACCGACUUCACGCUCACCAAGCAGCCUGCAGCCGGAGGACUUCGCCACCUACUACUGCCAGCAG CACUACACCACGCCCCCCACCUUCGGGCAGGGCACCAAGGUGGAGAUCAAGCGGACCGUGGCCGCCCCCA GCGUCUUCAUCUUCCCGCCCAGCGACGAGCAGCUGAAGUCGGGCACGGCCAGCGUGGUGUGCCUCCUGAA CAACUUCUACCCCCGCGAGGCGAAGGUCCAGUGGAAGGUGGACAACGCCCUGCAGAGCGGGAACAGCCAG GAGAGCGUGACCGAGCAGGACUCGAAGGACAGCACCUACAGCCUCAGCAGCACCCUGACGCUGAGCAAGG CCGACUACGAGAAGCACAAGGUCUACGCCUGCGAGGUGACCCACCAGGGGCUCUCGAGCCCCGUGACCAA GAGCUUCAACCGGGGCGAGUGCUGAAGCGCUGCCUUCUGCGGGGCUUGCCUUCUGGCCAUGCCCUUCUUC UCUCCCUUGCACCUGUACCUCUUGGUCUUUGAAUAAAGCCUGAGUAGGAAGGCGGCCGCUCGAGCAUGCA UCUAGA

The nucleic acid sequence for nucleotide sequence of the wild type CERTprotein is set forth in SEQ ID NO: 12:

(SEQ ID NO: 12) atgtcggata atcagagctg gaactcgtcg ggctcggagg aggatccaga gacggagtct gggccgcctg tggagcgctg cggggtcctc agtaagtgga caaactacat tcatgggtgg caggatcgtt gggtagtttt gaaaaataat gctctgagtt actacaaatc tgaagatgaa acagagtatg gctgcagagg atccatctgt cttagcaagg ctgtcatcac acctcacgat tttgatgaat gtcgatttga tattagtgta aatgatagtg tttggtatct tcgtgctcag gatccagatc atagacagca atggatagat gccattgaac agcacaagac tgaatctgga tatggatctg aatccagctt gcgtcgacat ggctcaatgg tgtccctggt gtctggagca agtggctact ctgcaacatc cacctcttca ttcaagaaag gccacagttt acgtgagaag ttggctgaaa tggaaacatt tagagacatc ttatgtagac aagttgacac gctacagaag tactttgatg cctgtgctga tgctgtctct aaggatgaac ttcaaaggga taaagtggta gaagatgatg aagatgactt tcctacaacg cgttctgatg gtgacttctt gcatagtacc aacggcaata aagaaaagtt atttccacat gtgacaccaa aaggaattaa tggtatagac tttaaagggg aagcgataac ttttaaagca actactgctg gaatccttgc aacactttct cattgtattg aactaatggt taaacgtgag gacagctggc agaagagact ggataaggaa actgagaaga aaagaagaac agaggaagca tataaaaatg caatgacaga acttaagaaa aaatcccact ttggaggacc agattatgaa gaaggcccta acagtctgat taatgaagaa gagttctttg atgctgttga agctgctctt gacagacaag ataaaataga agaacagtca cagagtgaaa aggtgagatt acattggcct acatccttgc cctctggaga tgccttttct tctgtgggga cacatagatt tgtccaaaag gttgaagaga tggtgcagaa ccacatgact tactcattac aggatgtagg cggagatgcc aattggcagt tggttgtaga agaaggagaa atgaaggtat acagaagaga agtagaagaa aatgggattg ttctggatcc tttaaaagct acccatgcag ttaaaggcgt cacaggacat gaagtctgca attatttctg gaatgttgac gttcgcaatg actgggaaac aactatagaa aactttcatg tggtggaaac attagctgat aatgcaatca tcatttatca aacacacaag agggtgtggc ctgcttctca gcgagacgta ttatatcttt ctgtcattcg aaagatacca gccttgactg aaaatgaccc tgaaacttgg atagtttgta atttttctgt ggatcatgac agtgctcctc taaacaaccg atgtgtccgt gccaaaataa atgttgctat gatttgtcaa accttggtaa gcccaccaga gggaaaccag gaaattagca gggacaacat tctatgcaag attacatatg tagctaatgt gaaccctgga ggatgggcac cagcctcagt gttaagggca gtggcaaagc gagagtatcc taaatttcta aaacgtttta cttcttacgt ccaagaaaaa actgcaggaa agcctatttt gttctag

The protein sequence for the wild type CERT protein is set forth in SEQID NO: 13:

(SEQ ID NO: 13) Met Ser Asp Asn Gin Ser Trp Asn Ser Ser Gly Ser Glu Glu Asp Pro Glu Thr Glu Ser Gly Pro Pro Val Glu Arg Cys Gly Val Leu Ser Lys Trp Thr Asn Tyr Ile His Gly Trp Gin Asp Arg Trp Val Val Leu Lys Asn Asn Ala Leu Ser Tyr Tyr Lys Ser Glu Asp Glu Thr Glu Tyr Gly Cys Arg Gly Ser Ile Cys Leu Ser Lys Ala Val Ile Thr Pro His Asp Phe Asp Glu Cys Arg Phe Asp Ile Ser Val Asn Asp Ser Val Trp Tyr Leu Arg Ala Gin Asp Pro Asp His Arg Gin Gin Trp Ile Asp Ala Ile Glu Gin His Lys Thr Glu Ser Gly Tyr Gly Ser Glu Ser Ser Leu Arg Arg His Gly Ser Met Val Ser Leu Val Ser Gly Ala Ser Gly Tyr Ser Ala Thr Ser Thr Ser Ser Phe Lys Lys Gly His Ser Leu Arg Glu Lys Leu Ala Glu Met Glu Thr Phe Arg Asp Ile Leu Cys Arg Gin Val Asp Thr Leu Gin Lys Tyr Phe Asp Ala Cys Ala Asp Ala Val Ser Lys Asp Glu Leu Gin Arg Asp Lys Val Val Glu Asp Asp Glu Asp Asp Phe Pro Thr Thr Arg Ser Asp Gly Asp Phe Leu His Ser Thr Asn Gly Asn Lys Glu Lys Leu Phe Pro His Val Thr Pro Lys Gly Ile Asn Gly Ile Asp Phe Lys Gly Glu Ala Ile Thr Phe Lys Ala Thr Thr Ala Gly Ile Leu Ala Thr Leu Ser His Cys Ile Glu Leu Met Val Lys Arg Glu Asp Ser Trp Gin Lys Arg Leu Asp Lys Glu Thr Glu Lys Lys Arg Arg Thr Glu Glu Ala Tyr Lys Asn Ala Met Thr Glu Leu Lys Lys Lys Ser His Phe Gly Gly Pro Asp Tyr Glu Glu Gly Pro Asn Ser Leu Ile Asn Glu Glu Glu Phe Phe Asp Ala Val Glu Ala Ala Leu Asp Arg Gin Asp Lys Ile Glu Glu Gin Ser Gin Ser Glu Lys Val Arg Leu His Trp Pro Thr Ser Leu Pro Ser Gly Asp Ala Phe Ser Ser Val Gly Thr His Arg Phe Val Gin Lys Val Glu Glu Met Val Gin Asn His Met Thr Tyr Ser Leu Gin Asp Val Gly Gly Asp Ala Asn Trp Gin Leu Val Val Glu Glu Gly Glu Met Lys Val Tyr Arg Arg Glu Val Glu Glu Asn Gly Ile Val Leu Asp Pro Leu Lys Ala Thr His Ala Val Lys Gly Val Thr Gly His Glu Val Cys Asn Tyr Phe Trp Asn Val Asp Val Arg Asn Asp Trp Glu Thr Thr Ile Glu Asn Phe His Val Val Glu Thr Leu Ala Asp Asn Ala Ile Ile Ile Tyr Gin Thr His Lys Arg Val Trp Pro Ala Ser Gin Arg Asp Val Leu Tyr Leu Ser Val Ile Arg Lys Ile Pro Ala Leu Thr Glu Asn Asp Pro Glu Thr Trp Ile Val Cys Asn Phe Ser Val Asp His Asp Ser Ala Pro Leu Asn Asn Arg Cys Val Arg Ala Lys Ile Asn Val Ala Met Ile Cys Gin Thr Leu Val Ser Pro Pro Glu Gly Asn  Gin Glu Ile Ser Arg

The nucleic acid sequence for the nucleotide sequence of the Ser132ACert mutant is set forth as SEQ ID NO: 14:

(SEQ ID NO: 14) atgtcggata atcagagctg gaactcgtcg ggctcggagg aggatccaga gacggagtct gggccgcctg tggagcgctg cggggtcctc agtaagtgga caaactacat tcatgggtgg caggatcgtt gggtagtttt gaaaaataat gctctgagtt actacaaatc tgaagatgaa acagagtatg gctgcagagg atccatctgt cttagcaagg ctgtcatcac acctcacgat tttgatgaat gtcgatttga tattagtgta aatgatagtg tttggtatct tcgtgctcag gatccagatc atagacagca atggatagat gccattgaac agcacaagac tgaatctgga tatggatctg aatccagctt gcgtcgacat ggcgcaatgg tgtccctggt gtctggagca agtggctact ctgcaacatc cacctcttca ttcaagaaag gccacagttt acgtgagaag ttggctgaaa tggaaacatt tagagacatc ttatgtagac aagttgacac gctacagaag tactttgatg cctgtgctga tgctgtctct aaggatgaac ttcaaaggga taaagtggta gaagatgatg aagatgactt tcctacaacg cgttctgatg gtgacttctt gcatagtacc aacggcaata aagaaaagtt atttccacat gtgacaccaa aaggaattaa tggtatagac tttaaagggg aagcgataac ttttaaagca actactgctg gaatccttgc aacactttct cattgtattg aactaatggt taaacgtgag gacagctggc agaagagact ggataaggaa actgagaaga aaagaagaac agaggaagca tataaaaatg caatgacaga acttaagaaa aaatcccact ttggaggacc agattatgaa gaaggcccta acagtctgat taatgaagaa gagttctttg atgctgttga agctgctctt gacagacaag ataaaataga agaacagtca cagagtgaaa aggtgagatt acattggcct acatccttgc cctctggaga tgccttttct tctgtgggga cacatagatt tgtccaaaag gttgaagaga tggtgcagaa ccacatgact tactcattac aggatgtagg cggagatgcc aattggcagt tggttgtaga agaaggagaa atgaaggtat acagaagaga agtagaagaa aatgggattg ttctggatcc tttaaaagct acccatgcag ttaaaggcgt cacaggacat gaagtctgca attatttctg gaatgttgac gttcgcaatg actgggaaac aactatagaa aactttcatg tggtggaaac attagctgat aatgcaatca tcatttatca aacacacaag agggtgtggc ctgcttctca gcgagacgta ttatatcttt ctgtcattcg aaagatacca gccttgactg aaaatgaccc tgaaacttgg atagtttgta atttttctgt ggatcatgac agtgctcctc taaacaaccg atgtgtccgt gccaaaataa atgttgctat gatttgtcaa accttggtaa gcccaccaga gggaaaccag gaaattagca gggacaacat tctatgcaag attacatatg tagctaatgt gaaccctgga ggatgggcac cagcctcagt gttaagggca gtggcaaagc gagagtatcc taaatttcta aaacgtttta cttcttacgt ccaagaaaaa actgcaggaa agcctatttt gttctag

The protein sequence of the Ser132A Cert mutant is set forth as SEQ IDNO. 15:

(SEQ ID NO: 15) Met Ser Asp Asn Gin Ser Trp Asn Ser Ser Gly Ser Glu Glu Asp Pro Glu Thr Glu Ser Gly Pro Pro Val Glu Arg Cys Gly Val Leu Ser Lys Trp Thr Asn Tyr Ile His Gly Trp Gin Asp Arg Trp Val Val Leu Lys Asn Asn Ala Leu Ser Tyr Tyr Lys Ser Glu Asp Glu Thr Glu Tyr Gly Cys Arg Gly Ser Ile Cys Leu Ser Lys Ala Val Ile Thr Pro His Asp Phe Asp Glu Cys Arg Phe Asp Ile Ser Val Asn Asp Ser Val Trp Tyr Leu Arg Ala Gin Asp Pro Asp His Arg Gin Gin Trp Ile Asp Ala Ile Glu Gin His Lys Thr Glu Ser Gly Tyr Gly Ser Glu Ser Ser Leu Arg Arg His Gly Ala Met Val Ser Leu Val Ser Gly Ala Ser Gly Tyr Ser Ala Thr Ser Thr Ser Ser Phe Lys Lys Gly His Ser Leu Arg Glu Lys Leu Ala Glu Met Glu Thr Phe Arg Asp Ile Leu Cys Arg Gin Val Asp Thr Leu Gin Lys Tyr Phe Asp Ala Cys Ala Asp Ala Val Ser Lys Asp Glu Leu Gin Arg Asp Lys Val Val Glu Asp Asp Glu Asp Asp Phe Pro Thr Thr Arg Ser Asp Gly Asp Phe Leu His Ser Thr Asn Gly Asn Lys Glu Lys Leu Phe Pro His Val Thr Pro Lys Gly Ile Asn Gly Ile Asp Phe Lys Gly Glu Ala Ile Thr Phe Lys Ala Thr Thr Ala Gly Ile Leu Ala Thr Leu Ser His Cys Ile Glu Leu Met Val Lys Arg Glu Asp Ser Trp Gin Lys Arg Leu Asp Lys Glu Thr Glu Lys Lys Arg Arg Thr Glu Glu Ala Tyr Lys Asn Ala Met Thr Glu Leu Lys Lys Lys Ser His Phe Gly Gly Pro Asp Tyr Glu Glu Gly Pro Asn Glu Phe Phe Asp Ala Val Glu Ala Ala Leu Asp Arg Gin Asp Lys Ile Glu Glu Gin Ser Gin Ser Glu Lys Val Arg Leu His Trp Pro Thr Ser Leu Pro Ser Gly Asp Ala Phe Ser Ser Val Gly Thr His Arg Phe Val Gin Lys Val Glu Glu Met Val Gin Asn His Met Thr Tyr Ser Leu Gin Asp Val Gly Gly Asp Ala Asn Trp Gin Leu Val Val Glu Glu Gly Glu Met Lys Val Tyr Arg Arg Glu Val Glu Glu Asn Gly Ile Val Leu Asp Pro Leu Lys Ala Thr His Ala Val Lys Gly Val Thr Gly His Glu Val Cys Asn Tyr Phe Trp Asn Val Asp Val Arg Asn Asp Trp Glu Thr Thr Ile Glu Asn Phe His Val Val Glu Thr Leu Ala Asp Asn Ala Ile Ile Ile Tyr Gin Thr His Lys Arg Val Trp Pro Ala Ser Gin Arg Asp Val Leu Tyr Leu Ser Val Ile Arg Lys Ile Pro Ala Leu Thr Glu Asn Asp Pro Glu Thr Trp Ile Val Cys Asn Phe Ser Val Asp His Asp Ser Ala Pro Leu Asn Asn Arg Cys Val Arg Ala Lys Ile Asn Val Ala Met Ile Cys Gin Thr Leu Val Ser Pro Pro Glu Gly Asn Gin Glu Ile Ser Arg Asp Asn Ile Leu Cys Lys Ile Thr Tyr Val Ala Asn Val Asn Pro Gly Gly Trp Ala Pro Ala Ser Val Leu Arg Ala Val Ala Lys Arg Glu Tyr Pro Lys Phe Leu Lys Arg Phe Thr Ser Tyr Val Gin Glu Lys Thr Ala Gly Lys Pro Ile Leu Phe

ELISA Detection of Human IgG Antibodies

FIG. 2 and FIG. 3 show an Enzyme-linked immunosorbent assay (ELISA) forHuman IgG from Chinese Hamster Ovary's (CHO) and Human Embryonic Kidney(HEK, HER-2 Negative) 293 cells transfected with human IgG modRNA,respectively. The Human Embryonic Kidney (HEK) 293 were grown in CD 293Medium with Supplement of L-Glutamine from Invitrogen until they reacheda confluence of 80-90%. The CHO cells were grown in CD CHO Medium withSupplement of L-Glutamine, Hypoxanthine and Thymidine. In FIG. 2, 2×10₆cells were transfected with 24 ug modRNA complexed with RNAiMax fromInvitrogen in a 75 cm₂ culture flask from Corning in 7 ml of medium. InFIG. 3, 80,000 cells were transfected with 1 ug modRNA complexed withRNAiMax from Invitrogen in a 24-well plate. The RNA:RNAiMAX complex wasformed by first incubating the RNA with CD 293 or CD CHO Medium in a 5×volumetric dilution for 10 minutes at room temperature. In a secondvial, RNAiMAX reagent was incubated with CD 293 Medium or CD CHO Mediumin a 10× volumetric dilution for 10 minutes at room temperature. The RNAvial was then mixed with the RNAiMAX vial and incubated for 20-30 atroom temperature before being added to the cells in a drop-wise fashion.In FIG. 2, the concentration of secreted human IgG in the culture mediumwas measured at 12, 24, 36 hours post-transfection. In FIG. 3, secretedhuman IgG was measured at 36 hours. The culture supernatants were storedat 4 degrees. Secretion of Trastuzumab from transfected Human EmbryonicKidney 293 cells was quantified using an ELISA kit from Abcam followingthe manufacturers recommended instructions. This data show that aHumanized IgG antibody (Trastuzumab) modRNA (SEQ ID NOs: 6 and 7) iscapable of being translated in Human Embryonic Kidney Cells and thatTrastuzumab is secreted out of the cells and released into theextracellular environment. Furthermore these data demonstrate thattransfection of cells with modRNA encoding Trastuzumab for theproduction of secreted protein can be scaled up to a bioreactor or largecell culture conditions.

Western Detection of modRNA Produced Human IgG Antibody.

FIG. 4 shows a Western Blot of CHO-K1 cells co-transfected with 1 μgeach of Heavy and Light Chain of Trastuzumab modRNA. In order to detecttranslation of protein product, cells were grown using standardprotocols in 24-well plates, and cell supernatants or cell lysates werecollected at 24 hours post-transfection and separated on a 12% SDS-Pagegel and transferred onto a nitrocellulose membrane using the iBlot byInvitrogen. After incubation with a rabbit polyclonal antibody to HumanIgG conjugated to DyLight® 594 (ab96904, abcam, Cambridge, Mass.) and asecondary goat polyclonal antibody to Rb IgG which was conjugated toalkaline phosphatase, the antibody was detected using Novex® alkalinephosphatase chromogenic substrate by Invitrogen.

Cell Immuno Staining of modRNA Produced Trastuzumab and Rituximab

FIG. 5 shows CHO-K1 cells co-transfected with 500 ng each of Heavy andLight Chain of Trastuzumab or Rituximab. Cells were grown in F-12KMedium from Gibco and 10% PBS. Cells were fixed with 4% paraformaldehydein PBS and permeabilized with 0.1% Triton X-100 in PBS for 5-10 minutesat room temperature. Cells were then washed 3× with room temperaturePBS. Trastuzumab and Rituximab staining was perfumed using rabbitpolyclonal antibody to Human IgG conjugated to DyLight® 594 (ab96904,abcam, Cambridge, Mass.) according to the manufacturer's recommendeddilutions. Nuclear DNA staining was performed with DAPI dye fromInvitrogen. The protein for Trastuzumab and Rituximab is translated andlocalized to the cytoplasm upon modRNA transfection. The pictures weretaken 13 hours post-transfection.

Binding Immunoblot Assay for modRNA Produced Trastuzumab and Rituximab

FIG. 6 shows a Binding Immunoblot detection assay for Trastuzumab andRituximab. Varying concentrations of the ErB2 peptide (ab40048, abcam,Cambridge, Mass.),

antigen for Trastuzumab and the CD20 peptide (ab97360, abcam, Cambridge,Mass.), antigen for Rituximab were run at varying concentrations (100ng/ul to 0 ng/μl) on a 12% SDS-Page gel and transferred onto a membraneusing the iBlot from Invitrogen. The membranes were incubated for 1 hourwith their respective cell supernatants from CHO-K1 cells co-transfectedwith 500 ng each of Heavy and Light Chain of Trastuzumab or Rituximab.The membranes were blocked with 1% BSA and a secondary anti-human IgGantibody conjugated to alkaline phosphatase (abcam, Cambridge, Mass.)was added. Antibody detection was conducted using the Novex® alkalinephosphatase chromogenic substrate by Invitrogen. This data show that ahumanized IgG antibodies generated from modRNA are capable ofrecognizing and binding to their respective antigens.

Cell Proliferation Assay

The SK-BR-3 cell line, an adherent cell line derived from a human breastadenocarcinoma, which overexpress the HER2/neu receptor can be used tocompare the antiproliferative properties of modRNA generatedTrastuzumab. Varying concentrations of purified Trastuzumab generatedfrom modRNA and trastuzumab can be added to cell cultures, and theireffects on cell growth can be assessed in triplicate cytotoxicity andviability assays.

SKOV-3 Tumor Model

The anti-cancer effects of modRNA generated Trastuzumab can bedetermined by consecutive injections of 1) modRNA Trastuzumab, 2)trastuzumab, and 3) modRNA Trastuzumab+modRNA GCSF over a period of 28days in SKOV-3 xenograft mice. The reduction in tumor growth size can bemonitored over time.

Example 4 Overexpression of Ceramide Transfer Protein to IncreaseTherapeutic Antibody Protein Production in Established CHO Cell Lines a)Batch Culture

An antibody producing CHO cell line (CHO DG44) secreting a humanizedtherapeutic IgG antibody is transfected a single time with lipidcationic delivery agent alone (control) or a synthetic mRNA transcriptencoding wild type ceramide transfer protein (CERT) or anon-phosphorylation competent Ser132A CERT mutant. CERT is an essentialcytosolic protein in mammalian cells that transfers the sphingolipidceramide from the endoplasmic reticulum to the Golgi complex where it isconverted to sphingomyelin (Hanada et al., 2003). Overexpression of CERTsignificantly enhances the transport of secreted proteins to the plasmamembrane and improves the production of proteins that are transportedvia the secretory pathway from eukaryotic cells thereby enhancingsecretion of proteins in the culture medium. Synthetic mRNA transcriptsare pre-mixed with a lipid cationic delivery agent at a 2-5:1carrier:RNA ratio. The initial seeding density is about 2×10₅ viablecells/mL. The synthetic mRNA transcript is delivered after initialculture seeding during the exponential culture growth phase to achieve afinal synthetic mRNA copy number between 10×10₂ and 10×10₃ per cell. Thebasal cell culture medium used for all phases of cell inoculumgeneration and for growth of cultures in bioreactors is modified CD-CHOmedium containing glutamine, sodium bicarbonate, insulin andmethotrexate. The pH of the medium is adjusted to 7.0 with 1 N HCl or 1NNaOH after addition of all components. Culture run times end on days 7,14, 21 or 28+. Production-level 50 L scale reactors (stainless steelreactor with two marine impellers) may be used and are scalableto >10,000 L stainless steel reactors (described in commonly-assignedpatent application U.S. Ser. No. 60/436,050, filed Dec. 23, 2002, andU.S. Ser. No. 10/740,645). A data acquisition system (Intellution Fix32) records temperature, pH, and dissolved oxygen (DO) throughout runs.Gas flows are controlled via rotometers. Air is sparged into the reactorvia a submerged frit (5 μm pore size) and through the reactor head spacefor C0₂ removal. Molecular oxygen is sparged through the same frit forDO control C0₂ is sparged through same flit as used for pH control.Samples of cells are removed from the reactor on a daily basis. A sampleused for cell counting is stained with trypan blue (Sigma, St. Louis,Mo.). Cell count and cell viability determination are performed viahemocytometry using a microscope. For analysis of metabolites,additional samples are centrifuged for 20 minutes at 2000 rpm (4° C.)for cell separation. Supernatant is analyzed for the followingparameters: titer, sialic acid, glucose, lactate, glutamine, glutamate,pH, pO₂, pCO₂, ammonia, and, optionally, lactate dehydrogenase (LDH).Additional back-up samples are frozen at −20° C. To measure secretedhumanized IgG antibody titers, supernatant is taken from seed-stockcultures of all stable cell pools, the IgG titer is determined by ELISAand divided by the mean number of cells to calculate the specificproductivity. The highest values are the cell pools with the Ser132ACERT mutant (SEQ ID No. 14), followed by wild type CERT (SEQ ID No. 12.In both, IgG expression is markedly enhanced compared to carrier-aloneor untransfected cells.

b) Continuous or Batch-Fed Culture

An antibody producing CHO cell line (CHO DG44) secreting humanized IgGantibody is transfected with lipid cationic delivery agent alone(control) or a synthetic mRNA transcript encoding wild type ceramidetransfer protein or a non-phosphorylation competent Ser132A CERT mutant.Synthetic mRNA transcripts are pre-mixed with a lipid cationic deliveryagent at a 2-5:1 carrier:RNA ratio. The initial seeding density wasabout 2×10₅ viable cell s/mL. Synthetic mRNA transcript is deliveredafter initial culture seeding during the exponential culture growthphase to achieve a final synthetic mRNA copy number between 10×10₂ and10×10₃ per cell. The basal cell culture medium used for all phases ofcell inoculum generation and for growth of cultures in bioreactors wasmodified CD-CHO medium containing lutamine, sodium bicarbonate, insulinand methotrexate. The pH of the medium is adjusted to 7.0 with 1 N HClor 1N NaOH after addition of all components. Bioreactors of 5 L scale(glass reactor with one marine impeller) are used to obtain maximum CERTprotein production and secreted humanized IgG antibody curves. Forcontinuous or fed-batch cultures, the culturing run time is increased bysupplementing the culture medium one or more times daily (orcontinuously) with fresh medium during the run. In the a continuous andfed-batch feeding regimens, the cultures receive feeding medium as acontinuously-supplied infusion, or other automated addition to theculture, in a timed, regulated, and/or programmed fashion so as toachieve and maintain the appropriate amount of synthetic mRNA:carrier inthe culture. The typical method is a feeding regimen of a once per daybolus feed with feeding medium containing synthetic mRNA: carrier oneach day of the culture run, from the beginning of the culture run tothe day of harvesting the cells. The daily feed amount is recorded onbatch sheets. Production-level 50 L scale reactors (stainless steelreactor with two murine impellers) were used and are scalable to >10,000L stainless steel reactors. A data acquisition system (Intellution Fix

32) record temperature, pH, and dissolved oxygen (DO) throughout runs.Gas flows are controlled via rotameters. Air is sparged into the reactorvia a submerged frit (5 μm pore size) and through the reactor head spacefor CO₂ removal. Molecular oxygen was sparged through the same frit forDO control. CO₂ is sparged through same frit as used for pH control.Samples of cells are removed from the reactor on a daily basis. A sampleused for cell counting is typically stained with trypan blue (Sigma, St.Louis, Mo.). Cell count and cell viability determination are performedvia hemocytometry using a microscope. For analysis of metabolites,additional samples are centrifuged for 20 minutes at 2000 rpm (4° C.)for cell separation. Supernatant is analyzed for the followingparameters titer, sialic acid, glucose, lactate, glutamine, glutamate,pH, pO₂, pCO₂, ammonia, and, optionally, lactate dehydrogenase (LDH).Additional back-up samples are frozen at −20° C. To measure secretedhumanized IgG antibody titers, supernatant is taken from seed-stockcultures of all stable cell pools, the IgG titer is determined by ELISAand divided by the mean number of cells to calculate the specificproductivity. The highest values are the cell pools with the Ser132ACERT mutant (SEQ ID NO: 14), followed by wild type CERT (SEQ ID NO: 10or 12). In both, IgG expression is markedly enhanced compared tocarrier-alone or untransfected cells.

EQUIVALENTS AND SCOPE

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments, described herein. The scope of the present disclosure isnot intended to be limited to the above Description, but rather is asset forth in the appended claims.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments in accordance with the disclosure described herein. Thescope of the present disclosure is not intended to be limited to theabove Description, but rather is as set forth in the appended claims.

In the claims articles such as “a,” “an,” and “the” may mean one or morethan one unless indicated to the contrary or otherwise evident from thecontext. Claims or descriptions that include “or” between one or moremembers of a group are considered satisfied if one, more than one, orall of the group members are present in, employed in, or otherwiserelevant to a given product or process unless indicated to the contraryor otherwise evident from the context. The disclosure includesembodiments in which exactly one member of the group is present in,employed in, or otherwise relevant to a given product or process. Thedisclosure includes embodiments in which more than one, or all of thegroup members are present in, employed in, or otherwise relevant to agiven product or process. Furthermore, it is to be understood that thedisclosure encompasses all variations, combinations, and permutations inwhich one or more limitations, elements, clauses, descriptive terms,etc., from one or more of the listed claims is

introduced into another claim. For example, any claim that is dependenton another claim can be modified to include one or more limitationsfound in any other claim that is dependent on the same base claim.Furthermore, where the claims recite a composition, it is to beunderstood that methods of using the composition for any of the purposesdisclosed herein are included, and methods of making the compositionaccording to any of the methods of making disclosed herein or othermethods known in the art are included, unless otherwise indicated orunless it would be evident to one of ordinary skill in the art that acontradiction or inconsistency would arise.

Where elements are presented as lists, e.g., in Markush group format, itis to be understood that each subgroup of the elements is alsodisclosed, and any element(s) can be removed from the group. It shouldit be understood that, in general, where the disclosure, or aspects ofthe disclosure, is/are referred to as comprising particular elements,features, etc., certain embodiments of the disclosure or aspects of thedisclosure consist, or consist essentially of, such elements, features,etc. For purposes of simplicity those embodiments have not beenspecifically set forth in haec verba herein. It is also noted that theterm “comprising” is intended to be open and permits the inclusion ofadditional elements or steps.

Where ranges are given, endpoints are included. Furthermore, it is to beunderstood that unless otherwise indicated or otherwise evident from thecontext and understanding of one of ordinary skill in the art, valuesthat are expressed as ranges can assume any specific value or subrangewithin the stated ranges in different embodiments of the disclosure, tothe tenth of the unit of the lower limit of the range, unless thecontext clearly dictates otherwise.

In addition, it is to be understood that any particular embodiment ofthe present disclosure that falls within the prior art may be explicitlyexcluded from any one or more of the claims. Since such embodiments aredeemed to be known to one of ordinary skill in the art, they may beexcluded even if the exclusion is not set forth explicitly herein. Anyparticular embodiment of the compositions of the disclosure (e.g., anyprotein; any nucleic acid; any method of production; any method of use;etc.) can be excluded from any one or more claims, for any reason,whether or not related to the existence of prior art.

All cited sources, for example, references, publications, databases,database entries, and art cited herein, are incorporated into thisapplication by reference, even if not expressly stated in the citation.In case of conflicting statements of a cited source and the instantapplication, the statement in the instant application shall control.

Other embodiments are in the claims.

What is claimed is:
 1. A method of producing a secreted immunoglobulinprotein of interest in a cell, comprising: i) providing a target cellcapable of protein translation; ii) introducing into the target cell acomposition comprising a first isolated nucleic acid comprising atranslatable region encoding the secreted immunoglobulin protein ofinterest and a nucleoside modification, under conditions such that theprotein of interest is produced in the cell, wherein the first isolatednucleic acid comprises a nucleotide sequence selected from the groupconsisting of: a) a nucleotide sequence comprising the nucleotidesequence of SEQ ID NO:5 or SEQ ID NO:7; and b) a nucleotide sequence atleast 95% identical to the nucleotide sequence of a), encoding the samepolypeptides as encoded by SEQ ID NO:5 or SEQ ID NO:7; iii)substantially purifying the secreted immunoglobulin; and iv) measuringthe binding of said secreted immunoglobulin to an antigen.
 2. The methodof claim 1, wherein the immunoglobulin is rituximab.
 3. The method ofclaim 1, wherein the target cell is a mammalian cell.
 4. The method ofclaim 1, wherein the first isolated nucleic acid is formulated in alipid-based delivery molecule.